In vivo acute changes in ATP and glucose concentrations in the testicles of prepubertal rats following unilateral torsion

Estudo In vivo das alterações agudas das concentrações de ATP e glicose induzidas por torção testicular unilateral nos testículos ipsilaterais do rato pré-púbere

Abstracts

Objetivo: Uma das causas mais freqüentes de dor escrotal intensa, de aparecimento súbito, em crianças e adolescentes, é a torção unilateral do testículo. O presente estudo tem como finalidade determinar as alterações agudas nas concentrações de ATP e glicose nos testículos submetidos à uma hora de isquemia induzida por torção testicular unilateral de 720°. Métodos: Sessenta ratos Wistar machos, pré-púberes, foram distribuídos em 2 grupos. O Grupo 1 (isquemia) incluiu 30 animais aleatoriamente distribuídos em 5 subgrupos e submetidos à isquemia testicular por rotação do cordão espermático em torno de seu eixo (720º). Após 60 minutos a torção foi desfeita e em intervalos variáveis (30 minutos, 1 h, 6 h e 24 h) procedeu-se à remoção dos testículos e coleta de amostra de sangue arterial. Os animais restantes (Grupo 2 - controle) foram igualmente distribuídos e submetidos aos mesmos procedimentos mas a distorção do testículo foi efetuada imediatamente após a torção. Resultados: As concentrações de ATP e glicose foram significativamente menores (P<0,05) no grupo isquêmico em comparação ao respectivo controle ao término do período de isquemia. Não foram observadas diferenças na glicemia, comparando-se os dois grupos. Conclusões: A torção testicular unilateral de 720°, por uma hora, leva a um decréscimo na perfusão com redução das concentrações tissulares de ATP e glicose no testículo ipsilateral. Estes achados permitem concluir que a isquemia reduz a capacidade de produção de ATP e a captação da glicose pelo testículo ipsilateral. Concentrações similares de ATP e glicose nos testículos ipsilaterais dos ratos submetidos à isquemia ou cirurgia simulada, após distorção, implicam em possível reversibilidade dessas alterações.

Torção do cordão espermático; Testículo; Isquemia; Traumatismo por reperfusão; Ratos

5 – ORIGINAL ARTICLE

IN VIVO ACUTE CHANGES IN ATP AND GLUCOSE CONCENTRATIONS IN THE TESTICLES OF PREPUBERTAL RATS FOLLOWING UNILATERAL TORSION¹

Sérgio Botelho Guimarães²

Paulo Roberto Leitão de Vasconcelos³

Guimarães SB, Vasconcelos PRL. In vivo acute changes in ATP and glucose concentrations in ipsilateral testicles of prepubertal rats following unilateral torsion. Acta Cir Bras [serial online] 2002 Mar-Apr;17(2). Available from URL: http://www.scielo.br/acb.

ABSTRACT: Objective: An experimental study has been conducted to investigate acute changes in testicular concentrations of ATP and glucose following one-hour unilateral 720° testicular torsion. Methods: Sixty prepubertal male Wistar rats were distributed into two groups. Group 1 (Ischemia) animals (n=30) were divided into 5 subgroups (n=6). All animals were subjected to right testicular torsion of 720° lasting one hour followed by detorsion. Testicles were collected and arterial blood samples taken from abdominal aorta at the end of ischemic period and 0.5, 1, 6 and 24 hours following detorsion. The remaining rats (Group 2 – Sham operated) were divided into 5 subgroups and submitted to a similar procedure but detorsion was performed immediately following torsion of the right testis. Results: ATP and glucose concentrations were significantly decreased (P<0.05) in ischemia group as compared to sham group at the end of ischemic period. There was no difference in glycemia when comparing ischemic versus sham-operated rats. Conclusions: Unilateral one-hour testicular torsion causes a decrease in ipsilateral testicular perfusion resulting in decreased testicular concentrations of glucose and ATP. This finding suggests that one-hour unilateral testicular torsion impairs testicular capacity to produce ATP (energy) due to glucose storage depletion and reduced glucose uptake. Similar concentrations of glucose and ATP in testicular tissues of rats submitted to ischemia (Group 1) and sham-operated rats (Group 2) following detorsion implies the possible reversibility of these changes.

KEY WORDS: Spermatic cord torsion. Testis. Ischemia. Reperfusion injury. Rats.

INTRODUCTION

The acutely painful scrotum is one of the most common surgical emergencies in the prepubertal boy¹. It is almost always caused by torsion of a testicular appendage or torsion of the testis². Children with torsion usually present with acute scrotal pain, nausea and vomiting.

Torsion of the testis impairs circulation and leads to ischemia. When a tissue is deprived of its oxygen supply the mitochondrial electron transport-oxidative phosphorylation sequence is inhibited, resulting in the decline of cellular levels of Adenosine-5'-triphosphate (ATP) and creatine phosphate. As cellular ATP levels diminish, anaerobic glycolysis is activated in an attempt to maintain normal cellular functions. Glycogen levels are rapidly depleted and lactic acid levels in the cytosol increase, reducing the intracellular pH³. Some tissues, such as kidney and testis are almost totally depending on glycolysis as a source of ATP because this tissue has relatively few mitochondria⁴. As the availability of glucose is reduced due to the circulation blockage imposed by torsion of the spermatic cord cellular concentration of ATP decreases due to a decreased rate in glycolysis. As soon as the offer of blood to the testis is resumed increase in ATP concentration would be expected.

Resumption and increase of blood perfusion flowing a period of ischemia may lead to further tissue damage due to increased production of oxygen-derived free radicals. These free radicals cause further cellular damage through the peroxidation of lipids in mitochondria and cell membranes⁵. Peroxidation of lipids in membranes changes membrane permeability and disrupts cell integrity secondary to membrane disruption. Unilateral testicular torsion of 720^o has been shown to cause an increase in lipid peroxidation in both testicles². Testicular blood flow decreases in the affected testis during torsion and increases significantly after detorsion⁶. Histological studies have demonstrated that animals between the ages of 35 and 50 days are highly susceptible to reproductive damage due to unilateral spermatic cord torsion⁷.

METHODS

Animal experiments carried out in the present study were performed in accordance with the International Norms for Biomedical Research in Animals (1990) and Federal Law n^o. 6.638 (Brazil, 08-May-1979)⁸. Sixty prepubertal male albino rats (Rattus norvegicus, variety Wistar), ages ranging between 35-42 days (mean: 40.05 days) and weighting between 97.0 and 170.0g (mean: 129.50 g) were obtained from the Faculty of Medicine, Federal University of Ceará. Animals were distributed at random into 2 large groups (Group 1 and Group 2) of 30 animals each.

Group 1 (Ischemia) 30 rats, divided in 5 subgroups, each comprising 6 rats.

Group 2 (Sham-operated) 30 rats, divided in 5 subgroups, each comprising 6 rats.

All surgical procedures were performed under general anesthesia with ethylic ether. Torsion, detorsion and orquiectomies were performed through slanting scrotal incisions. Rotating the right testicle 720° counterclockwise created torsion. Torsion lasted one hour and was maintained by transmesorchial suture in order to avoid parenchymal trauma⁹. No food was offered to the animals 12 hours before the surgical procedures and during the experiments. After each surgical intervention the skin incision was closed with 4-0 silk sutures. Rats were allowed to recover from anesthesia and were placed back in their cages; water ad libitum was offered until detorsion, when they were reanestetized. At the end of experiments, orchiectomies were performed and arterial blood samples were taken from abdominal aorta.

Group 1 rats were submitted to right testis torsion of 720° and orchidopexy as describe above. Detorsion of the right testis was carried out 1 hour later and the testis was replaced in the scrotal sac. Right testicles orquiectomies were performed and bood samples collected from abdominal aorta at the end of the ischemic period and 30 minutes, 1, 6 and 24 hours following detorsion of testis along with blood samples from abdominal aorta. Group 2 rats were submitted to right testis torsion of 720° in counterclockwise rotation, followed by immediate detorsion of the testicle. One hour later the testicles were removed and blood samples collected. Remaining animals were submitted to orquiectomy and collection of arterial blood from abdominal aorta 30 minutes, 1, 6 and 24 h later.

Following orchiectomies, all organs were freeze clamped in liquid nitrogen (minus 190^o Celsius), fine-grained and kept in individual glass vials containing HClO₄ (10%). The mixture was kept cold until centrifuged. The supernatant fraction (deproteinized supernatant) was neutralized and determinations of D-glucose and ATP concentrations performed. Heparinized blood samples were deproteinized in vials containing HClO₄ (10%) and kept cold until centrifuged. Following neutralization supernatant fractions were used as samples and glycemia measured.

Biochemical determinations

Blood and testicle glucose concentrations were measured according to the method described by SLEIN¹⁰ (1963). Testicular Adenosine-5'-triphosphate (ATP) concentrations were measured after the method described by LAMPRECHT & TRAUTSCHOLD¹¹ (1963).

All calculations were based upon different optical densities, measured at 340nm. Comparing the difference in absorbance of samples before and after the biochemical reactions take place, ATP and glucose concentrations were calculated as micromoles per gram of wet tissue.

Statistical analyses

Statistical analyses were carried out by comparing the means ± Standard Error (SEM) of each group. Non parametric Mann-Whitney test was performed using GraphPad Prism version 3.0 (GraphPad Software, USA). Statistical significance was accepted at p<0.05.

RESULTS

Intratesticular concentrations of ATP and glucose as well as blood concentrations of glucose are shown in Tables 1-3.

Torsion of testis (ischemia) lasting 1 hour induced a decrease (Table 1) in glucose tissue concentration of the right testis (rotated at 720°) of rats as compared to the concentrations found in the right testis of rats subjected to torsion of 720° and immediate detorsion followed by orquiectomy 1 hour later (0.21 ± 0.09 versus 2.26 ± 0.13 – P<0.05). Glucose concentrations in the testicles of ischemic rats were similar to those found in the testicles of sham-operated rats at all reperfusion times studied after detorsion (0.5h; 1h; 6h; 24h).

Ischemia induced a significant fall in the concentrations of ATP in the ischemic testicles of rats (Table 2) subjected to testicular 1 hour torsion as compared to the concentration found in tissue of the testicles of sham-operated rats (0,03 ± 0,02 versus 0,12 ± 0,03 – P < 0.05). ATP concentrations in the testicles of ischemic rats were similar to those found in the testicles of sham-operated rats at all reperfusion times studied after detorsion (0.5h; 1h; 6h; 24h).

Blood glucose concentrations were similar at all times studied (ischemia and reperfusion) in both Group 1 and Group 2 rats (Table 3).

DISCUSSION

Acute testicular torsion is a surgical emergency, which requires immediate intervention. The basic etiopathologic mechanism seems to be the sudden contraction of the cremasteric muscle that imposes an outward spiral rotation of the testicle¹². Trauma may also play a role in inducing torsion. The left testicle seems to be more affected than the right testicle. This may be related to anatomical variation, as the left cord is longer and tends to present an increased mobility, as compared to the right side.

The pathophysiology of the testicular torsion is related to ischemia imposed by torsion. Following the contortion of the cord venous return is severely impaired while the arterial blood flow is maintained. The resulting edema boosts venous compression leading to a vascular rupture and interstitial hemorrhage followed by generalized ischemia and testicular infarction¹³. The surgical outcome of the testicular torsion is related to the duration of ischemia as well as the degree of torsion. Beasley³ recommends surgical exploration of the scrotal sac if a definite diagnosis cannot be established at the end of 2 to 6 hours evaluation period. This latent period seems to be considerably shorter in research rat.

It has been shown that ipsilateral testicular blood flow does not return after 720o testicular torsion lasting more than 2 to 4 hours¹⁴. All experiment animals in this present study were subjected to one-hour ischemia. Decreased tissue concentrations of ATP and glucose in animal testicles demonstrated that 720o testicular torsion lead to testicular tissue anaerobic metabolism. The decrease in glucose concentration in the ischemic testicles of rats at the end of the ischemic period may be possibly due to decreased glucose uptake by rotated testicles. The fall in ATP concentrations at the end of 1 hour ischemia induced by torsion may be due to decreased availability of glucose and oxygen to the ischemic tissue which produced less energy (ATP production) as compared to the testicular tissue of rats subjected to sham operation. Reperfusion after detorsion of the testicles of rats from Group 1 led to ATP concentrations similar to those found in the testicles of sham-operated rats suggesting normalization of energy production during reperfusion.

Glucose availability to testicles in the reperfusion periods studied (0.5h; 1h; 6h and 24 h) was apparently the same as judged by the similar arterial concentrations of blood glucose in animals subjected to ischemia (Group 1) or sham-operation (Group 2).

CONCLUSIONS

This study demonstrates that unilateral one-hour testicular torsion causes a decrease in testicular perfusion of ipsilateral testis, leading to decreased testicular concentrations of glucose and ATP. This finding suggests that unilateral testicular torsion lasting one hour impairs testicular capacity to produce ATP (energy) due to glucose storage depletion and reduced glucose uptake. Similar concentrations of glucose and ATP in testicular tissues of rats submitted to ischemia (Group 1) and sham-operated rats (Group 2) following detorsion implies the possible reversibility of these changes.

Guimarães SB, Vasconcelos PRL. Estudo In vivo das alterações agudas das concentrações de ATP e glicose induzidas por torção testicular unilateral nos testículos ipsilaterais do rato pré-púbere. Acta Cir Bras [serial online] 2002 Mar-Abr;17(2). Disponível em URL: http://www.scielo.br/acb.

RESUMO:Objetivo: Uma das causas mais freqüentes de dor escrotal intensa, de aparecimento súbito, em crianças e adolescentes, é a torção unilateral do testículo. O presente estudo tem como finalidade determinar as alterações agudas nas concentrações de ATP e glicose nos testículos submetidos à uma hora de isquemia induzida por torção testicular unilateral de 720°. Métodos: Sessenta ratos Wistar machos, pré-púberes, foram distribuídos em 2 grupos. O Grupo 1 (isquemia) incluiu 30 animais aleatoriamente distribuídos em 5 subgrupos e submetidos à isquemia testicular por rotação do cordão espermático em torno de seu eixo (720^o). Após 60 minutos a torção foi desfeita e em intervalos variáveis (30 minutos, 1 h, 6 h e 24 h) procedeu-se à remoção dos testículos e coleta de amostra de sangue arterial. Os animais restantes (Grupo 2 – controle) foram igualmente distribuídos e submetidos aos mesmos procedimentos mas a distorção do testículo foi efetuada imediatamente após a torção. Resultados: As concentrações de ATP e glicose foram significativamente menores (P<0,05) no grupo isquêmico em comparação ao respectivo controle ao término do período de isquemia. Não foram observadas diferenças na glicemia, comparando-se os dois grupos. Conclusões: A torção testicular unilateral de 720°, por uma hora, leva a um decréscimo na perfusão com redução das concentrações tissulares de ATP e glicose no testículo ipsilateral. Estes achados permitem concluir que a isquemia reduz a capacidade de produção de ATP e a captação da glicose pelo testículo ipsilateral. Concentrações similares de ATP e glicose nos testículos ipsilaterais dos ratos submetidos à isquemia ou cirurgia simulada, após distorção, implicam em possível reversibilidade dessas alterações.

DESCRITORES: Torção do cordão espermático. Testículo. Isquemia. Traumatismo por reperfusão. Ratos.

Conflito de interesses: nenhum

Fontes de financiamento: nenhuma

Address for correspondence:

Sérgio Botelho Guimarães

Departamento de Cirurgia

Rua Prof. Costa Mendes, 1608/3^o andar

Fortaleza – Ceará

60430-140

Tel: [phone] Fax: [phone] [e-mail]

Data do recebimento: 18/01/2002

Data da revisão: 23/01/2002

Data da aprovação: 15/02/2002

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• 05 - Kagan VE. Lipid peroxidation in biomembranes. CRC Press: Boca Raton; 1988.
• 06 - Nguyen L, Lievano G, Ghosh L, Radhakrishnan J, Fornell L, John E. Effect of unilateral testicular torsion on blood flow and histology of contralateral testes. J Pediatr Surg 1999;34:680-3.
• 07 - Heindel RM, Pakyz RE, Reinking LN, Cosentino MJ. The effect of various degrees of unilateral spermatic cord torsion on fertility in the rat. J Urol 1990; 144:366-9.
• 08 - Brasil. Lei Federal n. 6.638, de 8 de maio de 1979. Estabelece normas para a prática didático-científica da vivisecção de animais e determina outras providências. In Brasil. Coleção de leis de 1979: atos legislativos do poder executivo. Leis de abril a junho. Brasília, Departamento de Imprensa Nacional, 1979. p 33-4.
• 09 - Ryan PC, Gorey TF, Fitzpatrick JM. Experimental testicular torsion: Fixation without parenchymal trauma. Eur Urol 1988;14:141-44.
• 10 - Slein MW. Determination with hexokinase and glucose-6-phospate dehydrogenase. In: Bergmeyer HU. Methods of enzymatic analysis. New York: Academic Press; 1963. p 117-23.
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• 12 - Sparks JP. Torsion of the testis. Ann R Coll Surg Engl 1971; 49:77-91.
• 13 - Cosentino MJ, Nishida M, Rabinowitz R, Cockett ATK. Histological changes occurring in the contralateral testicles of prepubertal rats subjected to various durations of unilateral spermatic cord torsion. J Urol 1985;133: 906-11.
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1

. From the Department of Surgery, Faculty of Medicine, Federal University of Ceará (UFC), Brazil.

2

. Professor Adjunto de Cirurgia, Vice-Coordenador de Graduação (Faculdade de Medicina – UFC) e Mestrando em Cirurgia Experimental do Departamento de Cirurgia, UFC, Fortaleza, CE.

3

. Professor Doutor (Doctor of Phylosophy), Coordenador do Curso de Pós-Graduação

stricto sensu em Cirurgia da Faculdade de Medicina (UFC), Fortaleza, CE.

Publication Dates

• Publication in this collection
  05 June 2002
• Date of issue
  Apr 2002

History

• Reviewed
  23 Jan 2002
• Received
  18 Jan 2002
• Accepted
  15 Feb 2002