Delayed onset, immunomodulation, and lifespan improvement of SOD1G93A mice after intravenous injection of human mesenchymal stem cells derived from adipose tissue.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective and progressive loss of motor neurons from the spinal cord, brain stem, and motor cortex. Although the hallmark of ALS is motor neuron degeneration, astrocytes, microglia, and T cells actively participate. Pharmacological treatment with riluzole has little effect on the lifespan of the patient. Thus, the development of new therapeutic strategies is of utmost importance. The objective of this study was to verify whether human mesenchymal stem cells (hMSCs) from adipose tissue have therapeutic potential in SOD1G93A transgenic mice. The treatment was carried out in the asymptomatic phase of the disease (10th week) by a single systemic application of ad-hMSCs (1 ×105 cells). The animals were sacrificed at the 14th week (the initial stage of symptoms) or the end-stage (ES) of the disease. The lumbar spinal cords were dissected and processed for Nissl staining (neuronal survival), immunohistochemistry (gliosis and synaptic preservation), and gene transcript expression (qRT-PCR). Behavioral analyses considering the onset of disease and its progression, neurological score, body weight, and motor control (rotarod test) started on the 10th week and were performed every three days until the ES of the disease. The results revealed that treatment with ad-hMSCs promoted greater neuronal survival (44%) than vehicle treatment. However, no effect was seen at the ES of the disease. Better structural preservation of the ventral horn in animals treated with ad-hMSCs was observed, together with decreased gliosis and greater synapse protection. In line with this, we found that the transcript levels of Hgf1 were upregulated in ad-hMSCs-treated mice. These results corroborate the behavioral data showing that ad-hMSCs had delayed motor deficits and reduced weight loss compared to vehicle animals. Additionally, cell therapy delayed the course of the disease and significantly improved survival by 20 days. Overall, our results indicate that treatment with ad-hMSCs has beneficial effects, enhancing neuronal survival and promoting a less degenerative neuronal microenvironment. Thus, this may be a potential therapy to improve the quality of life and to extend the lifespan of ALS patients.

Neuroprotection and gliosis attenuation by intravenous application of human mesenchymal stem cells (hMSC) following ventral root crush in mice.

Rupture and stretching of spinal roots are common incidents that take place in high-energy accidents. The proximal axotomy of motoneurons by crushing of ventral roots is directly related to the degeneration of half of the lesioned population within the first two weeks. Moreover, only a small percentage of surviving motoneurons can successfully achieve regeneration after such a proximal lesion, and new treatments are necessary to improve this scenario. In this sense, mesenchymal stem cells (MSC) are of great interest once they secrete a broad spectrum of bioactive molecules that are immunomodulatory and can restore the environment after a lesion. The present work aimed at studying the effects of human mesenchymal stem cells (hMSC) therapy after ventral root crush (VRC) in mice. We evaluated motoneuron survival, glial reaction, and synapse preservation at the ventral horn. For this purpose, C57BL/6 J were submitted to a crush procedure of L4 to L6 ventral roots and treated with a single intravenous injection of adipose-derived hMSC. Evaluation of the results was carried out at 7, 14, and 28 days after injury. Analysis of motoneuron survival and astrogliosis showed that hMSC treatment resulted in higher motoneuron preservation (motoneuron survival ipsi/contralateral ratio: VRC group = 53%, VRC + hMSC group = 66%; p < 0.01), combined with reduction of astrogliosis (ipsi/contralateral GFAP immunolabeling: VRC group = 470%, VRC + hMSC group = 250%; p < 0.001). The morphological classification and Sholl analysis of microglial activation revealed that hMSC treatment reduced type V and increased type II profiles, indicating an enhancement of surveying over activated microglial cells. The glial reactivity modulation directly influenced synaptic inputs in apposition to axotomized motoneurons. In the hMSC-treated group, synaptic maintenance was increased (ipsi/contralateral synaptophysin immunolabeling: VRC group = 53%, VRC + hMSC group = 64%; p < 0.05). Overall, the present data show that intravenous injection of hMSC has neuroprotective and anti-inflammatory effects, decreasing reactive astrogliosis, and microglial reaction. Also, such cell therapy results in motoneuron preservation, combined with significant maintenance of spinal cord circuits, in particular those related to the ventral horn.

Role of MHC-I Expression on Spinal Motoneuron Survival and Glial Reactions Following Ventral Root Crush in Mice.

Lesions to the CNS/PNS interface are especially severe, leading to elevated neuronal degeneration. In the present work, we establish the ventral root crush model for mice, and demonstrate the potential of such an approach, by analyzing injury evoked motoneuron loss, changes of synaptic coverage and concomitant glial responses in ß2-microglobulin knockout mice (ß2m KO). Young adult (8-12 weeks old) C57BL/6J (WT) and ß2m KO mice were submitted to a L4-L6 ventral roots crush. Neuronal survival revealed a time-dependent motoneuron-like cell loss, both in WT and ß2m KO mice. Along with neuronal loss, astrogliosis increased in WT mice, which was not observed in ß2m KO mice. Microglial responses were more pronounced during the acute phase after lesion and decreased over time, in WT and KO mice. At 7 days after lesion ß2m KO mice showed stronger Iba-1+ cell reaction. The synaptic inputs were reduced over time, but in ß2m KO, the synaptic loss was more prominent between 7 and 28 days after lesion. Taken together, the results herein demonstrate that ventral root crushing in mice provides robust data regarding neuronal loss and glial reaction. The retrograde reactions after injury were altered in the absence of functional MHC-I surface expression.

Neuroprotection and immunomodulation following intraspinal axotomy of motoneurons by treatment with adult mesenchymal stem cells.

BACKGROUND: Treatment of spinal cord injury is dependent on neuronal survival, appropriate synaptic circuit preservation, and inflammatory environment management. In this sense, mesenchymal stem cell (MSC) therapy is a promising tool that can reduce glial reaction and provide trophic factors to lesioned neurons. METHODS: Lewis adult female rats were submitted to a unilateral ventral funiculus cut at the spinal levels L4, L5, and L6. The animals were divided into the following groups: IA (intramedullary axotomy), IA + DMEM (Dulbecco's modified Eagle's medium), IA + FS (fibrin sealant), IA + MSC (106 cells), and IA + FS + MSC (106 cells). Seven days after injury, qPCR (n = 5) was performed to assess gene expression of VEGF, BDNF, iNOS2, arginase-1, TNF-α, IL-1ß, IL-6, IL-10, IL-4, IL-13, and TGF-ß. The cellular infiltrate at the lesion site was analyzed by hematoxylin-eosin (HE) staining and immunohistochemistry (IH) for Iba1 (microglia and macrophage marker) and arginase-1. Fourteen days after injury, spinal alpha motor neurons (MNs), evidenced by Nissl staining (n = 5), were counted. For the analysis of astrogliosis in spinal lamina IX and synaptic detachment around lesioned motor neurons (GAP-43-positive cells), anti-GFAP and anti-synaptophysin immunohistochemistry (n = 5) was performed, respectively. Twenty-eight days after IA, the gait of the animals was evaluated by the walking track test (CatWalk; n = 7). RESULTS: The site of injury displayed strong monocyte infiltration, containing arginase-1-expressing macrophages. The FS-treated group showed upregulation of iNOS2, arginase-1, proinflammatory cytokine (TNF-α and IL-1ß), and antiinflammatory cytokine (IL-10, IL-4, and IL-13) expression. Thus, FS enhanced early macrophage recruitment and proinflammatory cytokine expression, which accelerated inflammation. Rats treated with MSCs displayed high BDNF-positive immunolabeling, suggesting local delivery of this neurotrophin to lesioned motoneurons. This BDNF expression may have contributed to the increased neuronal survival and synapse preservation and decreased astrogliosis observed 14 days after injury. At 28 days after lesion, gait recovery was significantly improved in MSC-treated animals compared to that in the other groups. CONCLUSIONS: Overall, the present data demonstrate that MSC therapy is neuroprotective and, when associated with a FS, shifts the immune response to a proinflammatory profile.