Despite the advances in Raman spectroscopy, the clinical application of the technique is still limited and certain challenges must be overcome to enable clinical translation.
We provide an outlook on the future of the techniques in this area and what we believe is required to allow the potential of Raman spectroscopy to be achieved for clinical cancer diagnostics. Sloan-Dennison, S. Laing, D. Graham and K. Faulds, Chem. This article is licensed under a Creative Commons Attribution 3. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.
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This article is Open Access. Please wait while we load your content Something went wrong. Try again? S1 online. The expression of Bdnf, Gdnf , Ngf , and Vegf, which encode neurotrophic factors, and that of Tgfb and Tsg6, which encode anti-inflammatory factors, was evaluated. Real-time PCR analysis of neurotrophic and anti-inflammatory factor-associated genes. All rats showed recovery of motor function in these tests after SCI.
Rats in the rcMSC group exhibited more motor function recovery than those in the other groups. Effect of cell transplantation on motor function recovery. Results of the BBB scale a and inclined plane task score b. Effect of cell transplantation on electrophysiological recovery. Recovery rate of amplitude d. Cavity formation after spinal cord injury SCI. Staining of the cavity formed after SCI with hematoxylin and eosin. The graph in e shows the difference in the volume of cavities at 28 days after SCI between the three groups.
Effects of conditioned medium on the survival rate of stress-exposed NG cells. Survival rates of NG cells exposed to inflammatory stress a and oxidative stress b.
Herein, we investigated the effectiveness of rcMSCs in SCI and aimed to prove the recovery of motor function electrophysiologically and clarify the detailed underlying mechanism.
A previous study has shown that a part of the rat pyramidal tract runs on the posterior cord in the spinal cord Furthermore, several groups have used tcMEPs to study the motor pyramidal tract and neurological recovery in rat models of CNS injury 12 , 13 , 14 , Based on these previous findings, we concluded that tcMEPs can be used for behavioral evaluation and for the elucidation of the process of nerve function recovery in a rat model of posterior cord injury.
Longitudinal evaluation of tcMEPs is also required for behavioral evaluation. In our study, we recorded tcMEPs in a time-dependent manner. Specifically, in the rcMSC group, the amplitude of tcMEPs gradually recovered and correlated with the recovery of motor function.
These data suggested that rcMSCs transplantation resulted in the recovery of neural connections and regeneration of nerve fibers, playing a role in conduction 1. The recording of tcMEPs in cases of severe motor paralysis is challenging 16 , Accordingly, the waveform of tcMEP could not be recorded in the present study when the BBB scale and degree of the inclined plane test score were low, such as in the acute phase after SCI.
The appearance and improvement of the waveforms of tcMEP following motor function recovery are consistent with the observations of previous clinical reports 16 , Furthermore, as reported in humans, the existence of a measurable threshold for motor paralysis is also suspected in tcMEP recording of rats 16 , These cytokines induce an inflammatory response, increase vascular permeability, elevate the levels of reactive oxygen species, and finally cause cell death via apoptosis and necroptosis 22 , 23 , Cell death via necrosis and necroptosis is involved in the formation of cavities in the injured spinal cord tissue The effect of transplanting MSCs derived from bone marrow and adipose tissue on these inflammatory cytokines has also been reported in SCI models 21 , 27 , The role of oxidative and inflammatory stress in the injured tissues of the spinal cord has been reported The results of our in vitro experiments using stress-exposed NG cells are in accordance with those obtained in the in vivo experiments.
The survival rate of the NG cells was significantly higher in the rcMSC-CM group than that in the control group following exposure to oxidative or inflammatory stress. Bax is active upstream of the apoptotic cell death pathway, while caspase-3 is a downstream player 30 , Our results demonstrated that rcMSC-CM suppressed cell death under conditions of oxidative stress via the apoptotic pathways. Our results demonstrated that rcMSC-CM suppressed cell death under conditions of inflammatory stress via the necroptosis pathway.
These results suggest that rcMSCs may exert a stronger anti-apoptotic and ant-necroptosis effect via the neurotrophic factors than rbMSCs. Reports focusing on the role of the necroptosis pathway in the context of MSC transplantation are limited. Our results suggest that the rcMSC-transplantation affects both the necroptosis pathway and the apoptosis pathway in SCI tissue. In addition, the high expression of neurotrophic factors in rcMSCs can be expected to modulate the effects of transplantation in CNS disorders.
This study has certain limitations. Although behavioral and electrophysiological evaluations, such as the analysis of tcMEPs, were conducted, the sensory function could not be evaluated. In future studies, we plan to evaluate sensory disorders based on electrophysiological evaluations, such as somatosensory evoked potential, and study the therapeutic effect of rcMSCs in models of CNS injury.
We have provided novel evidence showing that cell-based therapy using rcMSCs results in excellent functional and electrophysiological recovery in an SCI rat model.
Bone marrow and cranial bones were seeded on culture dishes. Once cells had adhered to the bottom of the culture dish, approximately 5—7 days, nonadherent cells were eliminated by changing the culture medium, and the adherent cells were used as rbMSCs and rcMSCs.
Briefly, the cells were cultured in a mesenchymal stem cell osteogenic differentiation medium Promocell, Heidelberg, Germany and a mesenchymal stem cell adipogenic differentiation medium Promocell to induce osteogenic differentiation and adipogenic differentiation, respectively. Alizarin red S Sigma-Aldrich, St. Louis, United States and oil red O Wako Pure Chemical Industries, Osaka, Japan were used to stain the cells to confirm calcium deposition and lipid droplets, respectively.
The analysis of MSC-specific markers was performed as previously described 7 , In earlier studies, we reported the gene expression profile of rcMSCs 7 , In the present study, we added neurotrophic factors and anti-inflammatory markers to further investigate gene expression. Here, we investigated the expression of the genes encoding for rat brain-derived neurotrophic factor Bdnf , glial cell-derived neurotrophic factor Gdnf , nerve growth factor Ngf , and vascular endothelial growth factor Vegf.
The gene encoding glyceraldehydephosphate dehydrogenase Gapdh was used as the internal control to which the relative quantity of each mRNA was normalized. Adult female SD rats Charles River, Kanagawa, Japan , with a mean weight of g range, — g , were used for constructing an SCI model using the weight-dropping method 46 , The rats were anesthetized via the inhalation of 1.
A midline linear incision was made over the thoracic Th 9—11 spinous processes. Laminectomy was performed at Th An impactor rod was set on the surface of the spinal cord at Th10 and a cylindrical brass weight 10 g was dropped on the impactor. Following contusion, the skin was sutured to close the lesion. We divided the SCI rats into the following three groups according to the treatment received. Eleven rats were prepared in each group. MSCs 1. Considering the similar numbers of animals in published reports on rcMSCs 7 , 8 , we deemed our sample size sufficient to assess our model.
The BBB scale 47 , 48 , 49 , 50 and inclined plane task score 51 were used for determining the behavioral endpoints of the motor function recovery after SCI. In this study, behavioral analysis was performed immediately before the spinal injury and every day from pre-operation to days 1, 2, 3, 5, 7, 10, 14, 21, and 28 after surgery.
All motor functions were evaluated by an observer blinded to the group identities. Extended tcMEP was recorded using a bone-thinning technique To prepare for recording, the skulls of the rats were thinned with a diamond drill at the bregma and lambda positions.
Transcranial electrical stimulation was performed with a train of four stimuli administered across a total duration of 0. Onset amplitudes were recorded, and the recovery ratio of the amplitudes was calculated by dividing the amplitude at each point by that at pre-operation. We determined the cavity area ratio as an endpoint of histological recovery, as described in our previous paper 40 , 41 , 42 , 43 , 44 , 45 , Briefly, four weeks after transplantation, spinal cords were removed.
The cavity ratio of each group was calculated by dividing the cavity area by the total coronally resected spinal cord area The rats were anesthetized 24 h after MSC transplantation. Total RNA was extracted from the injured spinal cord segments the length of the segments was 2 mm, centered on the lesion site. RNA extraction and reverse transcription were performed as described above. Gapdh was used as an internal control.
MSC-CM was prepared as previously described 7 , 8. NG cells were exposed to oxidative and inflammatory stress to evaluate the neuroprotective effects of rbMSCs and rcMSCs, as previously described 7.
NG cells were seeded on culture dishes Sumitomo Bakelite Co. The cells were collected 24 h after exposure to stress. The remaining cells were collected to evaluate the survival rate and mRNA expression of NG cells. TaqMan gene primers for expression assays used in this study are listed in Supplementary Table S3 online. Muniswami, D. Motor recovery after transplantation of bone marrow mesenchymal stem cells in rat models of spinal cord injury. Pittenger, M. Multilineage potential of adult human mesenchymal stem cells.
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