Bone marrow stromal cells attenuate injury-induced changes in galanin, NPY and NPY Y1-receptor expression after a sciatic nerve constriction☆
Introduction
Single ligature nerve constriction (SLNC) is a newly developed animal model for the study of neuropathic pain (Brumovsky et al., 2004, Musolino et al., 2007, Coronel et al., 2008). Animals subjected to a sciatic nerve SLNC develop both mechanical (Brumovsky et al., 2004, Musolino et al., 2007) and thermal (Musolino et al., 2007) allodynia within 3 days of the lesion, and the allodynic responses are observed even 56 days after injury (Musolino et al., 2007). In this model, there are also dramatic phenotypic changes in primary afferent neurons, including changes in the expression of some neuropeptides involved in pain modulation (Brumovsky et al., 2004, Coronel et al., 2008). Thus, SLNC of the sciatic nerve results in a marked ipsilateral increase in neuropeptides galanin (Coronel et al., 2008) and tyrosine (NPY) (Brumovsky et al., 2004) like immunoreactivities (LIs) in lumbar dorsal root ganglia (DRGs), with a parallel decrease in the number of neurons expressing the NPY Y1-receptor (Y1R) (Brumovsky et al., 2004).
NPY and galanin have been shown to participate in the modulation of neuropathic pain, although the exact role they play has not yet been elucidated. Thus, proalgesic as well as analgesic actions have been described for both (Liu and Hökfelt, 2002, Holmes et al., 2005, Wiesenfeld-Hallin et al., 2005, Brumovsky et al., 2007, Gibbs et al., 2007, Smith et al., 2007). One factor that contributes to their complex action is the variety of receptors to which they respectively bind (Larhammar, 1996, Iismaa and Shine, 1999, Brain and Cox, 2006, Lang et al., 2007). In the case of NPY, the Y1R appears to be the subtype through which this neuropeptide exerts its analgesic effects (Xu et al., 1999, Naveilhan et al., 2001, Gibbs et al., 2007, Smith et al., 2007), while Y2R has been suggested to mediate NPY proalgesic actions (Brumovsky et al., 2007, Gibbs et al., 2007).
Bone marrow stromal cells (MSCs), also known as mesenchymal stem cells, have a well documented role in providing the appropiate microenvironment within the bone marrow which supports the tightly regulated process of hematopoiesis (Bianco et al., 2001, Short et al., 2003). It has recently been demonstrated that MSCs participate in the regeneration process that is activated following several types of lesion of the nervous system, contributing to the animals’ functional recovery (Chen et al., 2001, Lu et al., 2001, Cuevas et al., 2004, Mahmood et al., 2004). Thus, local MSC implantation in the distal stump of the transected rat sciatic nerve promotes functional recovery assessed by the walking track test (Cuevas et al., 2004). Also, in an animal model of cerebral ischemia, the intravenous administration of MSCs results in the selective engraftment of the cells in the ischemic hemisphere and in a significant recovery of the somatosensory behavior (Chen et al., 2001). Finally, MSCs administered intravenously to rats subjected to a traumatic brain injury preferentially migrate into the injured hemisphere, where they increase the expression of growth factors (Mahmood et al., 2004) and improve functional recovery (Lu et al., 2001, Mahmood et al., 2004).
We have recently shown that when MSCs are injected into the ipsilateral lumbar 4 (L4) DRGs of animals subjected to a sciatic nerve SLNC, these cells selectively migrate to the other lumbar ganglia affected by the lesion (ipsilateral L3, L5 and L6) (Coronel et al., 2006). In the ganglia where homing occurs, MSCs acquire a striking perineuronal localization, resembling glial/satellite cells (Coronel et al., 2006). This characteristic distribution, acquired in an active and time-dependent fashion, suggests an association with a specific role in the injured nervous tissue. In fact, MSC administration prevents the generation of mechanical allodynia and reduces the number of allodynic responses to cold stimuli (Musolino et al., 2007).
In this work, we have investigated the potential mechanisms involved in the reduction of neuropathic pain-related behaviors observed after MSC administration. For this purpose, we have analyzed by immunohistochemistry the expression of galanin, NPY and the Y1R in DRG neurons from animals subjected to a sciatic nerve SLNC and MSC intraganglionic administration.
Section snippets
Animals
Adult Sprague-Dawley male rats (200–300 g, Fucal, Buenos Aires, Argentina) were maintained in a 12 h light-cycle, with water and food ad libitum. All the experiments performed in this study were approved by the local Ethical Committee from the Department of Bioethics of the School of Biomedical Sciences from Austral University, and were carried out in accordance to the policy of the Society for Neuroscience and the International Association for the Study of Pain for the use of animals in pain
Results
Microscopical analysis (Fig. 1) and subsequent quantification (Fig. 2) of galanin-, NPY- and Y1R-immunoreactive (IR) neuronal profiles showed extensive changes in the expression of these markers in the ipsilateral L4–5 DRGs from animals with a sciatic nerve constriction. The intraganglionic injection of PBS or BNMCs did not modify this pattern of expression. However, MSC administration partially prevented the injury-induced changes in galanin, NPY and Y1R expression in both L4 and L5 DRGs.
Discussion
These results show that the changes observed in galanin, NPY and Y1R expression in primary afferent neurons after a sciatic nerve SLNC can be partially prevented by MSC administration. This effect cannot be reproduced after PBS or BNMC administration.
It is well known, that peripheral nerve injury induces drastic phenotypic changes in primary afferent neurons, involving a great variety of molecules among which are the neurotransmitters galanin and NPY. In normal conditions, only a few galanin (
Acknowledgements
We are grateful to Norma Alejandra Chasseing for her generous advice during the course of the experiments and to Silvina Ruffolo, Germán Ruffolo and Guillermo Gastón for their skilful technical assistance.
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Sources of support: Austral University, PICTO-CRUP 30930, Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina (CONICET) and Fundación Alberto Roemmers.