V. CONCLUSION
10. Star Pattern Recognition Analysis Using Plasma and Brain Samples
The levels of each of the 24 FFA in the MCAo and MCAo + hMSC groups were normalized to the corresponding mean values from the control group. These normalized values were used to create star graphs composed of 24 rays, so that the differences in the mean values among the control, MCAo, and MCAo + hMSC groups were presented clearly. Visual star patterns were produced for plasma and brain samples.
III. RESULTS
1. Transplanted hMSCs were Detected in the Ischemic Border Zone
TTC staining result showed infarcts were mostly located within the ipsilateral cortex and striatum (Figure 2A). Cells derived from intravenously delivered hMSCs were identified in host rat brains using the human specific antibody NuMA (Fig 2B). Transplanted hMSCs survived and were distributed throughout the ischemic damaged brain of recipient rats.
Approximately 5% (45 x 103 to 50 x 103) of NuMA-positive MSCs had migrated into the ischemic tissue 4 days after the transplantation of hMSCs into the MCAo model. These results are consistent with a previous report in a similar model (Li et al., 2002). A few NuMA-reactive cells were also observed in several areas of the ipsilateral hemisphere including the cortex and striatum (data not shown).
Fig. 2. TTC and hMSC staining. A. TTC-stained coronal brain sections.
Brain sections were stained with TTC to visualize the ischemic lesions one day after transient middle cerebral artery occlusion (MCAo). The cerebral infarct is identified as the white area, and the normal brain is stained red. Lesion development was limited to the territory of the MCA. B. hMSC labeled 5 days post MCAo. A marked increase in NuMA-positive MSCs can be seen in the ischemic border zone (scale bar: 20 μm).
2. Plasma FFA Composition in the Rat Model
The percentage composition of 24 FFA (μg/mL) was measured in plasma from eight
controls, six MCAo, and six MCAo + hMSC rats (Table 1). In the MCAo group compared to the control group, the levels of 15 FFA including caproic acid, were significantly increased, whereas the levels of palmitic acid, linoleic acid, arachidonic acid, and eicosenoic acids were significantly reduced (p<0.05 in all the cases). In the MCAo + hMSC group compared to the control group, the levels of 15 FFA including caproic acid, were significantly increased, whereas the levels of palmitic acid, linoleic acid, and arachidonic acid were significantly reduced. In the MCAo + hMSC group compared to the MCAo group, the levels of 16 FFA were decreased, but only the levels of myristic acid were significantly reduced down to a level similar to controls. In contrast, the levels of oleic-, stearic-, arachidonic-, eicosenoic-, eicosanoic-, docosapentaenoic-, and behenic acid were slightly increased in the MCAo + hMSC group.
Plotting the normalized FFA values from plasma samples in the tetracosagonal shape was very informative; it showed clearly the elevation of FFA in multiples (ranging from 0.72 to 1.60) of the control mean values (Figure 3). The levels of palmitic acid (No. 9), linoleic acid (No. 11), arachidonic acid (No. 14), and eicosenoic acid (No. 16) were lower in both the MCAo and MCAo + hMSC groups compared to the control group (Figure 3A, B). The amount of myristic acid (No. 7) in the plasma of the MCAo group was much greater than that of the MCAo + hMSC group (Figure 3C).
Table 1. The percentage composition of 24 FFAs found in rat plasma.
Fig. 3. Using star symbol plats overview of serum metabolism changes. Star symbol plots of sham, tMCA+PBS, and tMCAo + hMSC rat based on the mean percentage composition of each of the 24 free fatty acids in plasma, normalized to the corresponding mean control values. (A) control vs. MCAo, (B) control vs. MCAo + hMSC, and (C) MCAo vs. MCAo + hMSC. Ray numbers correspond to numbers in Table 1. * P<0.05,
**0.001<P<0.05, ***P<0.001
3. Brain FFA Composition in the Rat Model
In the brain, the levels of 24 FFA (ng/mg) were measured (Table 2), but the level of decenoic acid was lower than the quantification limit of the calibration curve and, therefore, the amount could not be determined. In the MCAo group compared to the control group, the levels of palmitic acid and oleic acid were significantly reduced, while the levels of myristic-, linoleic-, stearic-, eicosenoic-, eicosanoic-, docsapentaenoic-, erucic-, and behenic acid were significantly increased.
In the MCAo + hMSC group compared to the control group, the levels of 19 FFA were increased of which stearic-, eicosanoic-, docsapentaenoic-, erucic-, and behenic acid were significant. In contrast, the levels of palmitoleic-, palmitic-, and oleic acids were slightly reduced. In the MCAo + hMSC group compared to the MCAo group, the levels of myristic-, 1inoleic-, and eicosenoic acid were significantly reduced.
FFA levels in brain samples in MCAo and MCAo + hMSC groups were normalized to the corresponding mean control values showed elevation of FFA in multiples (ranging from 0.93 to 1.51) of control values (Figure 4). In the MCAo group, myristic acid was the most abundant, followed by linoleic acid (No. 11), whereas in the MCAo + hMSC group, eicosanoic acid (No. 17) was the most abundant, followed by behenic acid (No. 21) compared to the control group (Figure 4A, B). The levels of myristic acid (No. 7) and
Table 2. The percentage composition of 24 FFAs found in brain tissues.
a Valued shown are means ± standard deviations for the percentage composition by each FFA concentration (ng/mg). b Student’s t-test comparing the mean values of the MCAo group and MCAo + hMSC group with those of the control. c Student’s t-test comparing the mean values of the MCAo and MCAo + hMSC groups. N.D.: not determined.
Fig. 4. Using star symbol plats overview of brain metabolism changes. Star symbol plots of sham control, tMCAo+PBS, and tMCAo + hMSC rat based on the mean percentage composition of 23 free fatty acids in the brain, normalized to the corresponding mean control values. (A) control vs. MCAo, (B) control vs. MCAo + hMSC, and (C) MCAo vs. MCAo + hMSC. Ray numbers correspond to numbers in Table 1. * P<0.05, **0.001<P<0.05,
***P<0.001
IV. DISCUSSION
This is the first demonstration of changes in the composition of FFAs in the plasma and brains of rats with cerebral ischemia following transplantation of hMSC. There is increasing evidence that hMSC promote functional recovery in animal models of ischemic stroke.
Several mechanisms have been suggested by which hMSC facilitate recovery from stroke;
(a) replacement of infracted tissue by true differentiation, spontaneous cell fusion, or both, and (b) upregulation of endogenous recovery mechanism (neurogenesis or neuronal plasticity) related to the production of trophic factors released by the hMSC. My present data suggest another candidate mechanism by which hMSC facilitates recovery from stroke;
elevated plasma or tissue levels of several FFA normalized after hMSC transplantation.
Generally, deficiencies in essential PUFA may be caused by an inflammatory reaction involving excessive oxidation, diminished antioxidant defenses, cellular damage, high rates of cell division, and cytokine-mediated reactions (Das, 2006; Fritsche, 2006; Spector, 2001).
In particular, excessive amounts of oxygen radicals stimulate lipid peroxidation and the depletion of essential PUFAs, and this disturbs metabolic pathways during MCAo.
Compared to the control group, the significant increase in endogenously synthesized saturated FFAs (except for palmitic acid) and monounsaturated FFAs in the plasma of the MCAo and the MCAo + hMSC groups may be presumed to be compensatory to the deficiency of available PUFAs (Table 1, Figure 3). Moreover, the significant decrease in linoleic acid and arachidonic acid may explain the PUFA deficiency. In the MCAo + hMSC group, saturated FFAs, which have cholesterol-increasing effects, and monounsaturated
FFAs were slightly reduced compared to the MCAo group, with some exceptions (eicosenoic-, eicosanoic-, and behenic acid). This reduction has been reported to lower cholesterol and have a neuroprotective effect because the antioxidant properties play a role in the defense mechanisms against oxidative stress (Wang et al., 2006a). However, there was no significant difference in the levels of PUFAs in the MCAo and MCAo + hMSC groups.
The n-6 and n-3 PUFAs present in the brain are not synthesized de novo in humans, and must be either obtained from the diet or synthesized in the brain from linoleic acid (as the n-6 PUFA precursor) and γ -linolenic acid (as the n-3 PUFA precursor) supplied in plasma (Spector, 2001). In the MCAo group, the increased levels of linoleic acid and arachidonic acid may mediate and regulate inflammation, and this may explain the inflammatory reaction associated with MCAo. Moreover, these findings suggest that the brain obtained n-6 PUFAs, including linoleic acid, from plasma for a neuroprotective effect and to maintain brain function.
In the brain, the amount of the n-6 PUFA linoleic acid in the MCAo group was much higher than in the control group, whereas the levels in the MCAo + hMSC group were not different from those in the control group. This suggests that the disturbance of PUFA metabolism by MCAo may be restored to a normal state after transplantation of hMSCs.
Furthermore, this finding may explain the therapeutic amerlioration of the MCAo lesion by
the saturated and monounsaturated fatty acids that were increased in the MCAo group compared with the control group were reduced in the MCAo + hMSC group compared with the MCAo group. Also, the saturated and monounsaturated fatty acids that were reduced in the MCAo group compared to the control groups, were increased in the MCAo + hMSC group. These findings suggest that changes in the levels of FFA may occur to compensate for the deficiency of available PUFAs, compared to those in the control group. The levels of FFA may recover to normal levels after hMSC transplantation into MCAo rats. Therefore, the efficiency of stem cell therapy may increase by using PUFAs for the prevention of ischemic stroke.
The normalized values of the 24 FFA in the MCAo and MCAo + hMSC groups were more informative than the raw values because they expressed the elevation of the FFA levels in multiples of the mean control values. The overall size of the MCAo group in the star symbol plots was larger than that of the MCAo + hMSC group, i.e., the levels of FFA were generally greater in the MCAo group (Figure 3, 4). The two star patterns were similar, except for the levels of lauric acid (No. 5) and myristic acid (No. 7) in plasma samples and of myristic acid (No. 7) and linoleic acid (No. 11) in brain samples. Thus, the two groups could be distinguished from each other. The control mean served well as the control pattern for the MCAo and MCAo + hMSC groups and enabled the visual comparison of the two groups with the control group. Star symbol plots drawn based on these 24 variables were very useful for the visual pattern recognition of each group. The mean star plots representing the MCAo, MCAo + hMSC, and control groups were clearly distinguishable from one another.
Therefore, it may be possible to use biochemical monitoring of FFAs to follow remitting and
relapsing conditions, and to monitor the therapeutic efficacy of hMSC transplantation following MCAo.
Several limitations deserve to be mentioned. First, the FFA levels were not measured serially in the present study. Serial assessment of FFA levels is needed to test the time-related impact of ischemia per se and hMSC transplantation. Second, my present study focused on the changes in the FFA levels after stroke and hMSC transplatation; the biochemical mechanisms by which hMSC controls FFA levels are out of scope of this study.
Further studies concerning the link between stem cell biology are warranted.
V. CONCLUSION
In this study, metabolomic approach has provided insights into understanding the complexity of biochemical and physiological events that occur in ischemic brain injury and the therapeutic effects of MSCs in stroke. This method may provide a clinical tool for the biochemical monitoring of the therapeutic effects of hMSC transplantation in patients with various ischemic conditions. A better understanding of FFA metabolism after MSC transplantation could lead to the future development of effective strategies for stem cell therapy in cerebral ischemia.
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- 국문요약 –
허혈성 뇌졸중 쥐에서의 인간 중간엽 줄기세포를 이식 후 치료효과의 분석
아주대학교 대학원의학과 이 문 옥
(지 도 교 수: 이 광)
허혈성 뇌졸중은 뇌로 전달되는 혈관이 막힘으로 치명적인 뇌기능의 손실을 초래하며, 성인에게 신체적 결함을 일으키는 가장 큰 원인 중에 하나이다. 최근 세포대체 요법으로 주목을 받고 있는 줄기세포는 질병으로 인해 손상된 조직이 회복되기 위한 생물학적인 대체 세포를 제공한다는 측면에서 뇌졸중 치료에 새로운 가능성을 제시하고 있다. 본 연구에서는 뇌졸중 쥐에 인간 유래 중간엽 줄기세포 (Mesenchymal stem cells, MSCs)을 정맥으로 주입함으로써 줄기세포의 치료효과 및 그 기전을 관찰하려고 한다.
실험 I: 뇌졸중 환자와 같이 임상치료에 자가이식이 가능한 MSC 는 체외 배양으로 세포 수를 확장하여 이용한다. 하지만 배양횟수가 신경재생과
관찰하였다. 일부 hMSCs 는 신경세포로 분화되는 것을 관찰되었으며, 또한 신경 영양인자로 알려진 glial cell-line-derived neurotrophic factor(GDNF), nerve growth factor(NGF), vascular endothelial growth factor(VEGF), hepatocyte growth factor(HGF) 등의 영양인자는 초기 passage 의 MSC 를 처리한 뇌에서 matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) 분석을 이용하여 세 그룹간 발현 정도가 다른 14개의 단백
Synaptosomal-associated protein 25(SNAP-25)와 transitional endoplasmic reticulum ATPase 와 같은 단백질은 hMSCs 처리한 군에서 정상 수준으로 회 복 되였는데 이런 단백질은 신경세포의 손상이나 apoptosis 에 관여 하는 것으
로 알려져 있다. 이러한 프로티오믹스 프로필링 연구는 향후 뇌졸중 치료에 있어 electrophoresis; 프로티오믹스; 지방산; Gas chromatography-mass
spectrometry.