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  • 1. Pathology – Research and Practice 207 (2011) 775–779 Contents lists available at SciVerse ScienceDirect Pathology – Research and Practice journal homepage: articleThe ameliorating effect of dantrolene on the morphology of urinary bladder inspinal cord injured ratsBruno Torres ∗ , Rogéria Serakides, Fátima Caldeira, Mardelene Gomes, Eliane MeloDepartment of Veterinary Medicine and Surgery, School of Veterinary, Federal University of Minas Gerais, Belo Horizonte, MG, Brazila r t i c l e i n f o a b s t r a c tArticle history: In animal models of spinal cord injury (SCI), the urinary bladder can undergo significant structural andReceived 9 March 2011 physiological alterations. Dantrolene has been shown to be neuroprotective by reducing neuronal apo-Received in revised form 12 October 2011 ptosis after SCI. Furthermore, in addition to its anti-inflammatory and antioxidant properties, it appearsAccepted 12 October 2011 to have a beneficial action on voiding, once this drug acts on the external urethral sphincter relaxation. In the present study, we investigated the effects of dantrolene on urinary bladder injury that followsKeywords: experimental SCI. Forty-six male Wistar rats were laminectomized at T13, and a compressive traumaDantrolene was performed to induce SCI. After euthanasia, the urinary bladder was removed for gross and histologi-Spinal cord injuryUrinary bladder injury cal evaluation. Traumatized animals showed urinary retention with severe hemorrhagic cystitis. InjuredRat animals treated with dantrolene had less bladder hemorrhage and inflammatory infiltrate than those treated with placebo (p < 0.05). Our results demonstrate that dantrolene may protect against urinary bladder lesions that follow SCI. Treating spinal cord-injured patients with this agent may be a promis- ing additional therapeutic strategy to alleviate the accompanying inflammatory process. The results of the current study show that dantrolene has protective effects on spinal cord contusion-induced urinary bladder injury. The impaired integrity of bladder morphology was ameliorated by dantrolene treatment. © 2011 Elsevier GmbH. All rights reserved.Introduction therapeutic intervention. The development of any form of phar- macological therapy that can reduce or alleviate even some of the Spinal cord injury (SCI) produces primary damage at the injured adverse outcomes associated with SCI has proven difficult due tosite that is followed by a delayed secondary lesion extending the complexity of the injury [4,8,14,39]. Functional recovery ofrostrocaudally, leading to progressive tissue destruction. The neu- the lower urinary tract is important in patients with SCI to elim-rodegeneration induced by trauma is characterized by interruption inate devastating urinary problems and improve quality of lifeof ascending and descending axons, loss of neurons and glia and [23,25,28,29].demielinization, resulting in motor, sensory and autonomic func- Dantrolene is a drug that inhibits the ryanodine receptor Ca2+tional deficits [7,13,27,28]. The SCI alters the complex neural channels (RyR) located on the sarco-endoplasmatic reticulum incircuits that contribute to the coordinate activity of the bladder and skeletal muscle (RyR1) and neuronal cells (RyR3) [21,46]. It blocksexternal urethral sphincter (EUS) and causes significant alterations calcium-induced calcium release from intracellular Ca2+ stores,in lower urinary tract function. Spinal lesions above the lum- preventing cytosolic Ca2+ overload [12,21,42]. Clinically, dantro-bosacral level lead to inefficient voiding because the EUS contracts, lene is used as muscle relaxant and in the treatment of malignantwhile the bladder is contracting (detrusor-sphincter dyssynergia), hyperthermia [24,31]. It has been shown to possess antioxidantwhich impedes voiding, leads to large residual urine volume and [9,41] and anti-inflammatory [11,19] properties.bladder over distension, predisposing to inflammation and hemor- Previous investigations have assessed its neuroprotectiverhagic interstitial cystitis [2,18,25,28]. effects in several models of ischemic and traumatic brain injury In recent years, much attention has been focused on sec- [16,26,34,35,43], and traumatic [38], ischemia/reperfusion [22] andondary injury of SCI, which is an important potential target for compressive model of SCI [40]. Moreover, there are evidences that this drug acts on the relaxation of the skeletal muscle of EUS, thereby reducing the resistance to bladder voiding and help- ing to control micturition [17,20,37]. However, to the best of our ∗ Corresponding author at: Escola de Veterinária da UFMG, Avenida Antônio Car- knowledge, there are no reports of the effect of dantrolene on thelos 6627, Caixa Postal 567, Campus da UFMG, CEP 30123-970 Belo Horizonte, MG,Brazil. Tel.: +55 31 34092266; fax: +55 31 34092230. morphology of the urinary bladder after SCI. Therefore, the aim E-mail address: (B. Torres). of the current study was to investigate the potential effects of0344-0338/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.doi:10.1016/j.prp.2011.10.004
  • 2. 776 B. Torres et al. / Pathology – Research and Practice 207 (2011) 775–779dantrolene on urinary bladder injury that follows SCI in trauma- 10% phosphate-buffered formalin (pH 7.4) and fragments weretized rats. processed by the routine technique of embedding in paraffin. Four-micron sections were stained by hematoxylin–eosin (HE)Materials and methods technique for morphological evaluation under light microscopy. The bladder morphometry was performed to quantify amount of This study was approved and performed in agreement with the inflammatory infiltrate and hemorrhage in eight fields per ani-Ethical Principles in Animal Experimentation, adopted by the Ethics mal, using a 121-point graticule (40× objective). The results wereCommittee in Animal Experimentation from Federal University of expressed as percentage of lesion per field in each group.Minas Gerais (CETEA/UFMG, protocol no. 059/03). Statistical analysisAnimals and surgical procedure All data collected were analyzed using Prism 5 for Win- Forty-six male Wistar rats aged 12 weeks and weighing dows (GraphPad Software. La Jolla, CA, USA). Data from the320–350 g were used in this study. Rats were kept under a urinary bladders were evaluated for statistical significance using12/12 h light-dark cycle for 14 days of acclimation with commer- Kruskal–Wallis test with a post hoc Dunn’s multiple comparison.cial rodent food and water ad libitum. Pre-anesthetic medication These data were presented as mean ± SD of the percentage ofwas performed with tramadol (2 mg/kg, orally) and induction and lesions values. In all analyses, p value ≤ 0.05 was considered sta-maintenance was carried out with isoflurane administered by mask tistically a semi-opened system. The animals were positioned in proneposition, prepared for aseptic surgery and received prophylac-tic antibiotic therapy with cephalothin (30 mg/kg, intravenous). ResultsSkin and subcutaneous tissue were incised in the dorsal midlineextending from T6 to L1, the paravertebral muscles dissected and Gross and light microscopy findingslaminectomy of T13 was performed with the employment of apneumatic drill. After visualization of the spinal cord covered by The animals that underwent laminectomy alone (GII and GV)the intact dura, a compressive model of SCI was performed, as pre- showed neither gross nor histological changes in their urinaryviously described [1,5,36], using a weight of 70 g/cm loading to the bladders, while those subjected to SCI had bladder distensiondorsal surface of the spinal cord. Afterwards, the site was irrigated and hemorrhagic cystitis of varying intensity among the differentwith saline, the muscles approximated, and the reduced dead space groups. Histologically, at 32 h after SCI in GI and GIII, there wereand skin sutured using an unabsorbed suture. During anesthetic multifocal areas of hemorrhage in the muscle layers and laminarecovery, the animals were kept warm in a box heated approxi- propria of the urinary bladder, and mixed inflammatory infiltratemately to 37 ◦ C. They received tramadol (2 mg/kg, orally), every 8 h with macrophages, lymphocytes and neutrophils was also seen. Atfor three days. Abdominal massage was performed three times a eight days post-SCI, the bladders from GIV showed higher lesionday in all animals to assist with urination and defecation. intensity with inflammatory infiltrate and hemorrhage in all lay- ers when compared with rats that received dantrolene which hadTreatment inflammatory infiltrate predominantly consisting of macrophages in the lamina propria. Their morphological bladder features were The therapeutic protocol consisted of 10 mg/kg of dantrolene similar to those of non-traumatized animals (Fig. 1).(Cristália Lab. Itapira, SP, Brazil) diluted in 15 ml of water for injec- The quantification of these morphological findings showed thetion given in single dose, intraperitoneally 1 h after laminectomy. amount of hemorrhage and inflammatory infiltrate in the urinaryThe control groups received only water for injection as placebo bladder. At 32 h after SCI, the animals that received placebo (GI) hadgiven in single dose, intraperitoneally 1 h after laminectomy. significantly more hemorrhage (GI = 19%) than those that received dantrolene (GIII = 8.5%) (p < 0.01). At 32 h, the non-injured animalsExperimental groups showed no hemorrhage (GII = 0%). At eight days, the dantrolene- treated animals showed recovery from the hemorrhagic process The animals were randomly divided into six groups according (GVI = 0.6%) compared to the placebo-treated group (GIV = 15%)to the protocol of treatment and the time of euthanasia. GI (n = 7) (p < 0.001) and appeared not different when compared to the non-underwent laminectomy followed by SCI, treated with placebo and injured animals (GV = 0%) (p > 0.05) (Fig. 2). However, althougheuthanized after 32 h; GII (n = 7) underwent laminectomy alone, there was no significant difference in the inflammatory infiltratetreated with placebo and euthanized after 32 h; GIII (n = 8) under- between the traumatized groups at 32 h (GI = 17.7%; GIII = 12.4%), atwent laminectomy followed by SCI, treated with dantrolene and eight days the inflammatory infiltrate was smaller in animals whoeuthanized after 32 h; GIV (n = 8) underwent laminectomy followed received dantrolene (GVI = 6%) than in those who received placeboby SCI, treated with placebo and euthanized after eight days; GV (GIV = 16%) (p < 0.05) (Fig. 3).(n = 8) underwent laminectomy alone, treated with placebo andeuthanized after eight days; and GVI (n = 8) underwent laminec- Discussiontomy followed by SCI, treated with dantrolene and euthanized aftereight days. The study and its results were carried out by investiga- The results of the current study show that dantrolene has pro-tors who were blind to the experimental conditions. tective effects on spinal cord contusion-induced urinary bladder injury. The impaired integrity of bladder morphology was amelio-Gross and light microscopy rated by dantrolene treatment. Spinal cord injury produces severe deficits within the urogenital Rats were euthanized with an overdose of thiopental by system. The majority of these deficits are the result of disrup-intraperitoneal injection and necropsied to evaluate the integrity tion of supraspinal input to the spinal cord and reorganization ofof the urinary bladder. The harvests were always performed intraspinal circuitry in response to injury [18]. Micturition is medi-in the area visually more impaired. In animals without macro- ated by neural circuits that are located in the lumbosacral cordscopic lesions, the area was taken at random. They were fixed in [33]. Spinal injury above the lumbosacral level damages descending
  • 3. B. Torres et al. / Pathology – Research and Practice 207 (2011) 775–779 777Fig. 1. Light microscopy sections of urinary bladder of Wistar rats stained with H&E (A) normal bladder: transitional epithelium (EP), lamina propria (LP) and muscularis(TM). Normal urinary bladder of an animal from GII (non-injured – 32 h) – 115.3×, (B) Normal urinary bladder of an animal from GV (non-injured – eight days) – 58.5×;(C) Multifocal areas of hemorrhage (h) and inflammatory infiltrate (asterisk) in an animal from GI (placebo – 32 h) – 116.6×, (D) Multifocal areas of hemorrhage (h) andinflammatory infiltrate (asterisk) in an animal from GIII (dantrolene – 32 h) – 115.6×, (E) Inflammatory infiltrate (asterisk) and hemorrhage (h) in an animal from GIV (placebo– eight days) – 119.5×, (F) Urinary bladder in an animal from GVI (dantrolene – eight days) – 59.2× – showing recovery from hemorrhage process and less inflammatoryinfiltrate that the placebo group at eight days (GIV).pathways that normally coordinate somatic motor (via puden- is contracting (detrusor-sphincter dyssynergia), which impedesdal nerve) and parasympathetic control (via pelvic nerve) of the voiding, leads to large residual urine volume and bladder over-lower urinary tract, altering primary afferent pathways to the filling, predisposing to inflammation and hemorrhagic interstitiallumbosacral cord and, thus, impairing lower urinary tract function cystitis [2,18,25,28,45]. For all these reasons, patients with SCI[6,25]. are at higher risk for bacterial cystitis, chronic bacterial infec- In animal models of SCI, the urinary bladder can undergo tions within and under the uroepithelial layer, and bladder cancersignificant structural, physiological and molecular alterations. In [2,18,25].those animals, hematuria associated with a cellular inflamma- While there are some treatments for controlling detrusortory response and a breakdown of the uroepithelium lining the hyperreflexia, for example, antimuscarinic agents such as bethane-lumen of the bladder often occur as a result of bladder over- chol and neurotoxins such as capsaicin and resiniferatoxin,distention. This breakdown is initiated early after injury and is detrusor-sphincter dyssynergia remains difficult to manage with-characterized by a loss of transepithelial resistance and enhanced out catheterization or surgical interventions [29].permeability to both water and urea. These alterations result from To achieve therapeutic benefits on spinal cord-injured patients,inefficient voiding because the EUS contracts, while the bladder drugs that affect calcium homeostasis have been employed
  • 4. 778 B. Torres et al. / Pathology – Research and Practice 207 (2011) 775–779 the relaxation of the EUS has been previously reported and, con- sidering that voiding is one of the natural protective mechanisms of the urinary system, it is possible that the action of dantrolene on the sphincter has major roles in this process [17,20,32]. Importantly, our findings showed significant improvement of the damaged urinary bladder tissue in rats with SCI that received dantrolene. It is worth noting that, to rule out the possibility that pressure from the manual expression of urine was the source of hematuria, in this study the sham animals experienced bladder expression as well, as performed by Herrera et al. [18]. Even though we have not performed urodynamic tests, we believe that our macroscopic and histological findings, suggestive of an amelioration of a possible dyssynergia between bladder andFig. 2. Mean ± SD of the percentages of hemorrhage in urinary bladder of Wis- EUS, may be due to the direct action of the drug against the inflam-tar rats. GI (placebo – 32 h); GII (non-injured–32 h); GIII (dantrolene – 32 h); GIV matory process in the bladder wall and/or the direct action of the(placebo – 8 days); GV (non-injured–8 days) and GVI (dantrolene – 8 days) (*p ≤ 0.05; drug on the EUS skeletal muscle relaxation allowing a facilitated**p ≤ 0.01; ***p ≤ 0.001). voiding. Moreover, we can suggest the association of these possi- bilities.experimentally. Dantrolene, a ryanodine receptor antagonist, In summary, we demonstrate here, for the first time, thatinhibits Ca2+ efflux from the endoplasmic reticulum to the cytosol, systemically injected dantrolene ameliorates the urinary bladderresulting in a documented neuroprotective effect [3,21,30,46]. damage that follows SCI. These findings suggest that dantrolene Evidence for the neuroprotective effects of dantrolene via an may provide a promising additional therapeutic strategy for theantiapoptotic mechanism has been reported after experimental management of SCI and alleviate its consequences. Future investi-induction of neuronal death [26,31,34,43,44]. On the other hand, gation, such as concerning a long-term evaluation with urodynamicfew studies have been performed to investigate the effects of tests, must be done to elucidate the broad potential of this drug anddantrolene on SCI. Thorell et al. [38] examined the role of intracel- the exact pathway by which dantrolene promotes those benefits.lular calcium in mediating posttraumatic abnormalities in axonalconduction and demonstrated that dantrolene improved electro- Acknowledgmentsphysiological recovery in an in vitro model of compressive injuryto an isolated spinal cord dorsal column segment. 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