Neuroscience Letters 514 (2012) 102–105
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Metallothionein-II improves motor function recovery and increases spared tissue after spinal cord injury in rats Susana Arellano-Ruiz a,f , Camilo Rios a , Hermelinda Salgado-Ceballos b,c , Marisela Méndez-Armenta d , Leonardo del Valle-Mondragón e , Concepción Nava-Ruiz d , Marina Altagracia-Martínez f , Araceli Díaz-Ruiz a,∗ a
Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suarez S.S.A., Mexico Unidad de Investigación Médica en Enfermedades Neurológicas, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Mexico c Centro de Investigación del proyecto CAMINA A.C., Mexico d Laboratorio de Neuropatología, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suarez S.S.A., Mexico e Departamento de Farmacología, Instituto Nacional de Cardiología Ignacio Chávez S.S.A., Mexico f Maestría en Ciencias Farmacéuticas, Universidad Autónoma Metropolitana, Unidad Xochimilco, Mexico b
a r t i c l e
i n f o
Article history: Received 10 November 2011 Received in revised form 20 February 2012 Accepted 21 February 2012 Keywords: Metallothionein Spinal cord injury Neuroprotection BBB score
a b s t r a c t After spinal cord injury (SCI), a complex cascade of pathophysiological processes rapidly damages the nervous tissue. The initial damage spreads to the surrounding tissue by different mechanisms, including oxidative stress. We have recently reported that the induction of metallothionein (MT) protein is an endogenous rapid-response mechanism after SCI. Since the participation of MT in neuroprotective processes after SCI is still unknown, the aim of the present study was to evaluate the possible neuroprotective effect of exogenously administered MT-II during the acute phase after SCI in rats. Female Wistar rats weighing 200–250 g were submitted to spinal cord contusion by means of a computer-controlled device (NYU impactor). Rats received several doses of MT-II (3.2, 10 and 100 g) at 2 and 8 h after SCI. Results of the BBB scale were statistically analysed using an ANOVA of repeated-measures, followed by Tukey’s test. Among the three doses tested, only 10 and 100 g were able to signiﬁcantly increase (p < 0.05) BBB scale scores eight weeks after SCI from a mean of 7.88 in the control group, to means of 12.63 and 10.88 for the 10 and 100 g doses of MT-II, respectively. The amount of spared tissue was also higher in the groups treated with 10 and 100 g, as compared to the control group values. Results from the present study demonstrate a signiﬁcant neuroprotective effect of exogenously administered MT-II. Further studies are needed in order to characterize the mechanisms involved in this neuroprotective action. © 2012 Elsevier Ireland Ltd. All rights reserved.
Spinal cord injury (SCI) is characterized by two chronological events: the primary injury and the secondary injury. The primary injury is directly caused by the mechanical trauma  and leads to a complex cascade of pathophysiological processes known as secondary injury, expanding the site of damage soon after the injury and for a long time later, resulting in greater neurodegeneration and neurological dysfunction . Subsequently, auto-destructive mechanisms such as excitotoxicity , inﬂammation, oxidative stress  and cellular death by apoptosis or necrosis  are activated during secondary injury. Oxidative stress is an important mechanism involved in this process, where the antioxidant
∗ Corresponding author at: Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía, Ave. Insurgentes Sur No. 3877, Mexico City 14269, D.F., Mexico. Tel.: +52 55 55288036; fax: +52 55 54240808. E-mail address: [email protected]
(A. Díaz-Ruiz). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2012.02.068
defences are up-regulated in order to control the reactive oxygen species (ROS). In this context, metallothionein (MT) plays a role as antioxidant thiol-defence in the acute phase of SCI. MT is a family of low molecular weight (6–7 kDa) proteins with a high content of cysteine, and bound metal ions . The metal thiolate clusters (Scys –M–Scys ) exist in two separate globular domains, the ␣ and ␤ domains, which are linked by a small lysine-rich region; the ␣-domain in the C-terminus contains 11 cysteines and is able to bind four divalent or six monovalent metals, while the N-terminal domain contains 9 cysteines capable of binding three divalent or six monovalent metals . MT functions include the transport and storage of essential transition metals and detoxiﬁcation and protection against ROS , which are important mechanisms for the host defence response, immunoregulation, cell survival and brain repair . In spinal cord, the MT-III isoform has been localized in motor neurons , while MT-I and MT-II are expressed in astrocytes and microglia . Recently, we showed an increase, relative
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to sham-group values, in MT content at 4, 12 and 24 h, after SCI suggesting an important neuroprotective role of MT in the acute phase of this pathology . Several reports have demonstrated the participation of MT in neurodegenerative and traumatic diseases, where the presence of MT has been observed to diminish damage through anti-oxidative, anti-apoptotic and anti-inﬂammatory mechanisms . Although, Penkowa and Hidalgo demonstrated that MT administered exogenously could inhibit damage in a model of autoimmune encephalomyelitis in rats . Based on this information, we proposed to evaluate the neuroprotective effect of MT administered exogenously after a SCI by contusion in rats. Animal care and the protocols for animal use were approved by the Animals Ethics Committee of the National Institute of Neurology and Neurosurgery of Mexico. We used adult female Wistar rats (200–250 g) randomly assigned to one of four groups with SCI. Rats were housed two per cage with a 12 h light/dark cycle and free access to food and water. All animals were handled according to the National Institutes of Health (USA) Guidelines for the Care and Use of Laboratory Animals. The spinal cord contusion was performed under pentobarbital (50 mg/kg i.p.) anesthesia, following the method described by Basso et al. . An incision was made extending from the mid to low thoracic regions, followed by a laminectomy, including all T9, to expose the spinal cord. The NYU weight-drop device was used in order to produce the SCI by contusion. Thus, the spinal clamps were attached to the T8 and T10 spinous processes, a transducer was placed over the transverse process of T9, and the impact rod was centered above it. The rod was slowly lowered until it contacted the dura, which was determined by completion of a circuit that activated a tone. Then, the SCI was produced by dropping the 10-g rod from a distance of 25 mm. The surgical site was sutured in layers. Rats were allowed to recover from anesthetic and surgical procedures in an intensive care unit for small animals (Schoer Manufacturing Co., Kansas City, MO, USA). Rats were randomly assigned to one of the following four groups: one control group that was administered with vehicle only (saline solution, 0.9% NaCl) and three experimental groups (n = 36) that were exogenously administered with different doses of MT-II dissolved in saline solution (metallothionein II from rabbit liver, Sigma M9542, as Cd content ≤0.5% and Zn content 4–9%, and essentially free of total salt impurities), at i.p. doses of 3.2, 10 or 100 g, as reported by Penkowa and Hidalgo . Two and eight hours after SCI, the dose was repeated, in accordance with reports by Diaz-Ruiz et al. , showing the times for minimal values of endogenous MT after SCI. MT-II was selected on the basis of the stability of MT-II. MT-II half-life in adult animals is of 21 vs 33 h, while the half-life of MT-I is shorter. This may indicate that MT-I is more susceptible to degradation than MT-II . Motor function recovery was evaluated by the Basso–Bettie–Bresnahan (BBB) scale, an open-ﬁeld locomotor test for rats . In an open ﬁeld (120 cm × 120 cm), the behavior of animals was observed for 5 min by three individuals blinded to the treatment. The scale was designed to reﬂect progressive motor rating scores. In brief, the BBB scale involves closely monitoring limb movement, weight-bearing capability, coordinated and proper gait, movement at all joints of the hindlimb, full weight support and appropriate limb, body and tail position. Animals were evaluated 24 h after the SCI with the aim to conﬁrm the absence of movement and then once a week for eight weeks after the injury. Finally, the animals were perfused transcardially with 10% buffered formalin at the end of the study. Approximately 1 cm of the spinal cord at the T9 level was removed immediately and embedded in a 10 mm parafﬁn block. Longitudinal sections (10-m thick) were obtained and stained with Kl¨vver-Barrera technique in order to identify neurons and myelin ﬁbers. Scar tissue around cavities and tissue with abnormal architecture was considered to be damaged tissue. The spared spinal cord tissue surrounding the lesion
Fig. 1. Metallothionein II (MT-II) effects upon recovery of hind limb motor function after spinal cord contusion injury. Motor recovery as measured by open ﬁeld BBB score. Animals were evaluated 1 day (D1) after lesion and then weekly for two months. SCI: spinal cord contusion treated with vehicle (550 l of saline solution), SCI/MT3.2, SCI/MT 10 and SCI/MT 1000: spinal cord contusion and treatment with MT-II 3.2, 10 and 100 g, 2 and 8 h after damage. The results are expressed as means ± SEM of 8–9 animals per group and analyzed by repeated-measures ANOVA followed by Tukey’s test, *different from SCI group (p < 0.05).
was measured, and the results were expressed as mm2 of tissue, mean ± SEM from 8 to 9 animals per group. In order to have a comparable area for evaluation, the ependyma was taken as the point of reference, and the area with the most tissue destruction was considered to be the epicenter of the injury. Sample photographs were digitized using a computerized system equipped with IM 500 software and a 200 FX digital camera. Pathologic analysis was performed with an Image Database V.4.01 (Leica) and a CCD-IRIS Sony camera, using morphometric assessment. Signiﬁcant differences in BBB scores were determined using the repeated-measures ANOVA followed by Tukey’s test, while the spared tissue area was analyzed by using one-way ANOVA followed by Tukey’s test. All analyses were performed with SPSS 17.0 software. Differences were considered statistically signiﬁcant at p < 0.05. The motor function recovery results obtained from the evaluation over the course of 8 weeks using the BBB scale, which showed that animals treated with doses of 10 and 100 g, but not with dose of 3.2 g of MT-II, 2 and 8 h after the injury, displayed better functional recovery compared with animals in the control group treated just with vehicle. At the end of the study, the BBB scale mean value for motor function was 7.88 ± 1.28 for the control group, while for animals treated with the MT-II doses of 10 and 100 g was 12.63 ± 2.07 and 10.88 ± 1.90, respectively (p < 0.05). These results are shown in Fig. 1. A dose–response curve was generated and adjusted using the Hill’s equation to obtain K (ED50: effective dose) and Emax (maximum effect) values. This equation correlates the dose with the effect. The results were K = 1.1 and E = 46.2 (Fig. 2). Histological studies showed better preservation of the spinal cord tissue cytoarchitecture in animals treated with doses of 10 and 100 g of MT-II compared with animals from control group (Fig. 3). The preserved area of tissue was 5.32 ± 0.31 mm2 in control-group animals, which was signiﬁcant less than in the injured animals treated with MT-II at 10 and 100 g (6.62 ± 0.21 and 6.29 ± 0.31, respectively) (p < 0.05). Representative images are depicted in Fig. 4. Results from the present study demonstrate that the exogenous administration of MT-II at doses of 10 and 100 g, 2 and 8 h after
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Fig. 2. Graphic of dose–response curve shown the results of AUC of animals treated with different doses of MT-II (3.2, 10 and 100 g) administered 2 and 8 h after the injury and evaluated each week during 8 weeks. This results were adjusted with Hill equation, area = (E × Doses)/(K + Doses); K = 1.073 and E = 46.235.
SCI promotes signiﬁcant motor function recovery, which correlates with an increased amount of spared spinal cord tissue. These results could be explained due to the antioxidant effect of this protein. A common feature observed in many studies in vitro and in vivo is that MT-I and MT-II reduce cell death and brain damage [22,32]. The highly antioxidant capacity of MT (even greater than that of glutathione) is one of the mechanisms through which this cytoprotective effect is mediated . MT readily reacts with the hydroxyl, superoxide and nitric oxide radicals, and thiol groups of MT prevent peroxynitrite and anion peroxynitroso-compounds formation, rendering this protein a highly efﬁcient tool in antioxidant defence [2,21]. Studies in cultured cells modiﬁed to overexpress MT-I and MT-II have demonstrated a clear protection against oxidative stress
Fig. 4. Effect of metallothionein-II upon spared tissue area 8 weeks after spinal cord injury in rats. SCI: spinal cord contusion treated with vehicle (550 l of saline solution), SCI/MT3.2, SCI/MT 10 and SCI/MT 1000: spinal cord contusion and treatment with MT-II 3.2, 10 and 100 g 2 and 8 h after damage. The results are expressed as means ± SEM of 8–9 animals per group and analyzed by repeated-measures ANOVA followed by Tukey’s test. *Different from SCI group (p < 0.05).
conditions [19,33]. This anti-oxidant capacity has been evaluated in models of central nervous system damage, revealing diminished lipid peroxidation, and less tyrosine nitration generated after peroxynitrite formation [13,29]. However, the anti-inﬂammatory effect of MT in autoimmune diseases of the central nervous system, such as multiple sclerosis, has also been described in association with the inhibition of CD4+, in the presence of MT-I and MT-II . The anti-inﬂammatory capacity of MT-I and MT-II has been reported in experimental models of autoimmune encephalitis, with the effect being mediated by the inhibition of pro-inﬂammatory
Fig. 3. Micrographs of the site of injury of rats subjected to spinal cord contusion and sacriﬁced 8 weeks after damage, showing the methods from determination of spared spinal cord tissue in longitudinal section. (A) Representative micrographs of the epicenter of the lesion of animals injured and vehicle only, (B–D) rats with spinal cord injury and treated with MT-II 3.2, 10 and 100 g, 2 and 8 h after damage respectively. Luxol Fast-Blue stain.
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cytokines such as IL-6 and tumor necrosis factor ␣ (TNF␣), as shown by Penkowa and Hidalgo . This study also found that MT-I and MT-II reduce apoptosis associated with decreased TNF␣, an effect that was corroborated by Giralt et al. under different brain-damaging conditions . MT-I and MT-II may also inhibit apoptosis induced by 6-aminonicotinamide . Notably, the overexpression of MT-I and MT-II promotes axonal regeneration and neurite outgrowth . MT-I and MT-II may also function as neurotrophic factors when added directly to cultures of hippocampal and dopaminergic neurons, encouraging survival and promoting axonal growth . The results of a recent study to investigate the effect of MT in dorsal root ganglia injured in the presence or absence of exogenous MT showed that MT signiﬁcantly increased neuronal regeneration 16 h after injury . On the other hand, it is also likely that the antioxidant effect of MT administered only in the acute stage of injury is key to promote motor recovery and to increase the amount of spared tissue, as lipoperoxidative damage has been reported 24 h after SCI . In any case, it has been documented that neuroprotective therapies should be initiated at short times after SCI to regulate the secondary mechanisms of injury that started from a few minutes after the trauma (primary lesion) and that the success of certain drugs depends on the speed of administration [7,10]. The mechanism by which MT exerts its neuroprotective effect when administered exogenously has not been fully clariﬁed; however, the presence of MT in the extracellular space in rats with brain damage was recently demonstrated. As the half-life for MT-II is derived mainly from in vivo studies, it is not known which is the half-life of the injected protein. It might be the same upon cell internalization, but the remaining extracellular MT may be substantially ﬁltered by the kidney. It has been observed that astrocytes secrete MT, and this is internalized by neurons in a megalin receptor-dependent manner . This may be the mechanism through which the MT also exerts its effect after SCI and can be considered to be a potential neuroprotective strategy to prevent damage and to induce regeneration. Acknowledgment This work was supported by the CONACyT; contract grant number 70064
  
  
  
   
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