Administration of riluzole, tempol and mesenchymal stem cells in treatment of ALS in SOD1G93A mice

Author(s)

Gabriela Bortolança Chiarotto

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2018-01-26

Examining board members

Alexandre Leite Rodrigues de Oliveira; Rosalia Mendez Otero; Rui Seabra Ferreira Junior; Marcondes Cavalcante Franca Junior; Cláudia Vianna Maurer Morelli

• Advisor: Alexandre Leite Rodrigues de Oliveira

Abstract

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by the selective and progressive loss of motor neurons in the spinal cord, brain stem, and motor cortex. It results in progressive atrophy and consequent muscular paralysis, leading the patient to death, usually between 2 and 5 years after the onset of symptoms. Although its etiology is complex and little known, it is believed that, as in other neurodegenerative diseases, the underlying pathological mechanism of ALS is a set of cellular and biochemical alterations that end up triggering the degeneration of the motoneurons. Although the end result of the disease is neuronal death, it is known today that ALS is a non-autonomous disease with the active participation of astrocytes, microglia and T cells as protagonists of neuroinflammation, one of the main and most evident pathogenic mechanisms involved in the disease. Pharmacological treatment with riluzole is not curative and has little effect on the prolongation of patient survival. Thus, the development of new therapeutic strategies aimed at promoting a better quality of life for patients is of utmost importance. As an alternative, there is the prospect of the use of cyclic nitroxides such as tempol, since they are multifunctional antioxidants that present low toxicity in experimental animals. In addition to pharmacological therapies, the use of stem cells has been heavily investigated, seeking immunomodulatory and neuroprotective effects. The objective of this study was to verify if the interaction between riluzole, tempol and mesenchymal stem cells presents therapeutic potential in SOD1G93A transgenic mice. The treatments started in the asymptomatic phase of the disease (10th week). Treatment with riluzole (8mg/kg) and tempol (50mg/kg) was performed on alternate days until the 14th week and from there to the final stage of the disease, twice a week. On the other hand, the systemic application (1x105) of human mesenchymal stem cells (hMSC) derived from adipose tissue occurred only once. The animals were sacrificed at the 14th week (initial stage of symptoms - ISS) and at the end stage (ES) of the disease for sample collection and data analysis. The lumbar spinal cord of the animals was dissected and processed for the following techniques: Nissl staining for evaluation of neuronal survival, immunohistochemistry to evaluate astrogliosis, microgliosis and synaptic changes (ISS and ES); qRT-PCR to evaluate the expression of neurotrophic factors and pro-inflammatory cytokines (ISS) and transmission electron microscopy for the ultrastructural evaluation of the alpha motoneurons (ES). Behavioral analyzes considering the onset of disease, progression and survival of the animals through evaluation of the neurological score, body weight monitoring and Rotarod motor performance test, started on the 10th week and were performed every 3 days until the stage end of the disease. The results revealed that treatment with tempol and hMSC promoted greater neuronal survival (23% and 44%, respectively) in the disease ISS when compared to animals in the vehicle group. However, this effect was only evidenced in the ES of the disease in the group treated with tempol. The higher neuronal survival observed in the ISS was accompanied by a greater preservation of synaptic circuits in the ventral horn of the spinal cord of these animals. However, in the final stage, there was ~ 80% loss of synapses in all experimental groups when compared to non-transgenic animals (NTG; p <0.0001). The intense reactivity of astrocytes and microglia observed in vehicle animals, when compared to NTG, was reduced in the lumbar spinal cord of the animals treated with tempol, hMSC and hMSC + tempol, being evidenced in the hMSC group when purchased at the tempol. The neuroprotective effect of the treatments with tempol and hMSC under the inflammation were continuous, until the ES of the disease. In addition to the lower reactivity of glial cells, the groups treated with tempol and hMSC reduced the expression of proinflammatory cytokines (IL1beta and TNFalpha) and ~ 3x the expression of TGFbeta (anti-inflammatory) in the ISS, when compared with the vehicle group. All these added effects resulted in delayed motor deficit and reduced the weight loss of these animals when compared to vehicle animals. In addition, groups receiving cell therapy alone or in combination with tempol delayed the course of the disease, which significantly reflected an 18-day increase in survival of the animals. Together, our results indicate that treatment with tempol as well as cell therapy with hMSC have beneficial effects, enhancing neuronal survival, preserving synapses and decreasing glial cell reactivity during ALS progression, and reducing levels of proinflammatory cytokines in the spinal cord, and may be considered promising therapies to promote patients' quality of life and / or to extend their survival time (AU)

• FAPESP's process: 13/16168-8 - Administration of riluzole, tempol and mesenchymal stem cells in the treatment of ALS in SOD1 G93A mice
• Grantee: Gabriela Bortolança Chiarotto
• Support Opportunities: Scholarships in Brazil - Doctorate