Iba1+/NG2+ macrophage-like cells expressing a variety of neuroprotective factors ameliorate ischemic damage of the brain

In a transient 90-min middle cerebral artery occlusion (MCAO) model of rats, a large ischemic lesion is formed where macrophage-like cells massively accumulate, many of which express a macrophage marker, Iba1, and an oligodendrocyte progenitor cell marker, NG2 chondroitin sulfate proteoglycan (NG2); therefore, the cells were termed BINCs (Brain Iba1⁺/NG2⁺ Cells). A bone marrow transplantation experiment using green-fluorescent protein-transgenic rats showed that BINCs were derived from bone marrow. 5-Fluorouracil (5FU) injection at 2 days post reperfusion (2 dpr) markedly reduced the number of BINCs at 7 dpr, causing enlargement of necrotic volumes and frequent death of the rats. When isolated BINCs were transplanted into 5FU-aggravated ischemic lesion, the volume of the lesion was much reduced. Quantitative real-time RT-PCR showed that BINCs expressed mRNAs encoding bFGF, BMP2, BMP4, BMP7, GDNF, HGF, IGF-1, PDGF-A, and VEGF. In particular, BINCs expressed IGF-1 mRNA at a very high level. Immunohistochemical staining showed that IGF-1-expressing BINCs were found not only in rat but also human ischemic brain lesions. These results suggest that bone marrow-derived BINCs play a beneficial role in ischemic brain lesions, at least in part, through secretion of neuroprotective factors.     

The asparaginyl endopeptidase legumain after experimental stroke

Various proteases in the brain contribute to ischemic brain injury. We investigated the involvement of the asparaginyl endopeptidase legumain after experimental stroke. On the basis of gene array studies and in situ hybridizations, we observed an increase of legumain expression in the peri-infarct area of rats after transient occlusion of the middle cerebral artery (MCAO) for 120 mins with a maximum expression at 24 and 48 h. Immunohistochemical analyses revealed the expression of legumain in Iba1⁺ microglial cells and glial fibrillary acidic protein-positive astrocytes of the peri-infarct area in mice after MCAO. Post-stroke recovery was also studied in aged legumain-deficient mice (45 to 58 weeks old). Legumain-deficient mice did not show any differences in physiologic parameters compared with respective littermates before, during MCAO (45 mins), and the subsequent recovery period of 8 days. Moreover, legumain deficiency had no effect on mortality, infarct volume, and the neurologic deficit determined by the rotating pole test, a standardized grip strength test, and the pole test. However, a reduced number of invading CD74⁺ cells in the ischemic hemisphere indicates an involvement in post-stroke inflammation. We conclude that legumain is not essential for the functional deficit after MCAO but may be involved in mechanisms of immune cell invasion.     

Immune cell infiltration in malignant middle cerebral artery infarction: comparison with transient cerebral ischemia

We tested whether significant leukocyte infiltration occurs in a mouse model of permanent cerebral ischemia. C57BL6/J male mice underwent either permanent (3 or 24 hours) or transient (1 or 2 hours+22- to 23-hour reperfusion) middle cerebral artery occlusion (MCAO). Using flow cytometry, we observed ∼15,000 leukocytes (CD45^+high cells) in the ischemic hemisphere as early as 3 hours after permanent MCAO (pMCAO), comprising ∼40% lymphoid cells and ∼60% myeloid cells. Neutrophils were the predominant cell type entering the brain, and were increased to ∼5,000 as early as 3 hours after pMCAO. Several cell types (monocytes, macrophages, B lymphocytes, CD8⁺ T lymphocytes, and natural killer cells) were also increased at 3 hours to levels sustained for 24 hours, whereas others (CD4⁺ T cells, natural killer T cells, and dendritic cells) were unchanged at 3 hours, but were increased by 24 hours after pMCAO. Immunohistochemical analysis revealed that leukocytes typically had entered and widely dispersed throughout the parenchyma of the infarct within 3 hours. Moreover, compared with pMCAO, there were ∼50% fewer infiltrating leukocytes at 24 hours after transient MCAO (tMCAO), independent of infarct size. Microglial cell numbers were bilaterally increased in both models. These findings indicate that a profound infiltration of inflammatory cells occurs in the brain early after focal ischemia, especially without reperfusion.     

The KCa3.1 blocker TRAM-34 reduces infarction and neurological deficit in a rat model of ischemia/reperfusion stroke

Microglia and brain infiltrating macrophages significantly contribute to the secondary inflammatory damage in the wake of ischemic stroke. Here, we investigated whether inhibition of KCa3.1 (IKCa1/KCNN4), a calcium-activated K⁺ channel that is involved in microglia and macrophage activation and expression of which increases on microglia in the infarcted area, has beneficial effects in a rat model of ischemic stroke. Using an HPLC/MS assay, we first confirmed that our small molecule KCa3.1 blocker TRAM-34 effectively penetrates into the brain and achieves micromolar plasma and brain concentrations after intraperitoneal injection. Then, we subjected male Wistar rats to 90 minutes of middle cerebral artery occlusion (MCAO) and administered either vehicle or TRAM-34 (10 or 40 mg/kg intraperitoneally twice daily) for 7 days starting 12 hours after reperfusion. Both compound doses reduced infarct area by ∼50% as determined by hematoxylin & eosin staining on day 7 and the higher dose also significantly improved neurological deficit. We further observed a significant reduction in ED1⁺-activated microglia and TUNEL-positive neurons as well as increases in NeuN⁺ neurons in the infarcted hemisphere. Our findings suggest that KCa3.1 blockade constitutes an attractive approach for the treatment of ischemic stroke because it is still effective when initiated 12 hours after the insult.     

Regulatory T cells accumulate and proliferate in the ischemic hemisphere for up to 30 days after MCAO

Local and peripheral immune responses are activated after ischemic stroke. In our present study, we investigated the temporal distribution, location, induction, and function of regulatory T cells (Tregs) and the possible involvement of microglia, macrophages, and dendritic cells after middle cerebral artery occlusion (MCAO). C57BL/6J and Foxp3^EGFP transgenic mice were subjected to 30 minutes MCAO. On days 7, 14, and 30 after MCAO, Tregs and antigen presenting cells were analyzed using fluorescence activated cell sorting multicolor staining and immunohistochemistry. A strong accumulation of Tregs was observed on days 14 and 30 in the ischemic hemisphere accompanied by the elevated presence and activation of microglia. Dendritic cells and macrophages were found on each analyzed day. About 60% of Foxp3⁺ Tregs in ischemic hemispheres were positive for the proliferation marker Ki-67 on days 7 and 14 after MCAO. The transfer of naive CD4⁺ cells depleted of Foxp3⁺ Tregs into RAG1^−/− mice 1 day before MCAO did not lead to a de novo generation of Tregs 14 days after surgery. After depletion of CD25⁺ Tregs, no changes regarding neurologic outcome were detected. The sustained presence of Tregs in the brain after MCAO indicates a long-lasting immunological alteration and involvement of brain cells in immunoregulatory mechanisms.     

Transplantation of human mesenchymal stem cells promotes functional improvement and increased expression of neurotrophic factors in a rat focal cerebral ischemia model

Previous studies have suggested that intravenous transplantation of mesenchymal stem cells (MSCs) in rat ischemia models reduces ischemia‐induced brain damage. Here, we analyzed the expression of neurotrophic factors in transplanted human MSCs and host brain tissue in rat middle cerebral artery occlusion (MCAO) ischemia model. At 1 day after transient MCAO, 3 × 10⁶ immortalized human MSC line (B10) cells or PBS was intravenously transplanted. Behavioral tests, infarction volume, and B10 cell migration were investigated at 1, 3, 7, and 14 days after MCAO. The expression of endogenous (rat origin) and exogenous (human origin) neurotorphic factors and cytokines was evaluated by quantitative real‐time RT‐PCR and Western blot analysis. Compared with PBS controls, rats receiving MSC transplantation showed improved functional recovery and reduced brain infarction volume at 7 and 14 days after MCAO. In MSC‐transplanted brain, among many neurotrofic factors, only human insulin‐like growth factor 1 (IGF‐1) was detected in the core and ischemic border zone at 3 days after MCAO, whereas host cells expressed markedly higher neurotrophic factors (rat origin) than control rats, especially vascular endothelial growth factor (VEGF) at 3 days and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) at 7 days after MCAO. Intravenously transplanted human MSCs induced functional improvement, reduced infarct volume, and neuroprotection in ischemic rats, possibly by providing IGF‐1 and inducing VEGF, EGF, and bFGF neurotrophic factors in host brain. © 2009 Wiley‐Liss, Inc.     

小鼠真皮多能干细胞在缺血性损伤脑组织移植后的分布与分化

目的探讨小鼠真皮多能干细胞(SKP)经股静脉移植后在缺血性损伤脑组织中的分布及分化情况。方法分离培养绿色荧光蛋白转基因小鼠(C57BL/6-gfp)SKP,随机选取5只同基因型小鼠采用线栓法制作局灶性大脑中动脉栓塞(MCAO)模型,缺血2h后行再灌注,再灌注24h后将C57BL/6-gfp来源的SKP经小鼠股静脉输入动物模型体内,植入后第7天处死小鼠,作冷冻切片,采用免疫组织荧光染色法,检测SKP在脑缺血小鼠体内的分布及分化情况。结果移植SKP7d后,MCAO小鼠脑组织冷冻切片在荧光显微镜下可见移植的SKP主要分布在缺血灶周围,且这些细胞表达胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)。结论经股静脉移植的SKP主要分布在MCAO模型鼠脑缺血损伤区周围,并可向神经细胞分化。     

Knockout of silent information regulator 2 (SIRT2) preserves neurological function after experimental stroke in mice

Sirtuin-2 (Sirt2) is a member of the NAD⁺-dependent protein deacetylase family. Various members of the sirtuin class have been found to be involved in processes related to longevity, regulation of inflammation, and neuroprotection. Induction of Sirt2 mRNA was found in the whole hemisphere after experimental stroke in a recent screening approach. Moreover, Sirt2 protein is highly expressed in myelin-rich brain regions after stroke. To examine the effects of Sirt2 on ischemic stroke, we induced transient focal cerebral ischemia in adult male Sirt2-knockout and wild-type mice. Two stroke models with different occlusion times were applied: a severe ischemia (45 minutes of middle cerebral artery occlusion (MCAO)) and a mild one (15 minutes of MCAO), which was used to focus on subcortical infarcts. Neurological deficit was determined at 48 hours after 45 minutes of MCAO, and up to 7 days after induction of 15 minutes of cerebral ischemia. In contrast to recent data on Sirt1, Sirt2^−/− mice showed less neurological deficits in both models of experimental stroke, with the strongest manifestation after 48 hours of reperfusion. However, we did not observe a significant difference of stroke volumes or inflammatory cell count between Sirt2-deficient and wild-type mice. Thus we postulate that Sirt2 mediates myelin-dependent neuronal dysfunction during the early phase after ischemic stroke.     

Mild hypothermia combined with neural stem cell transplantation for hypoxic-ischemic encephalopathy： neuroprotective effects of combined therapy

Neural stem cell transplantation is a useful treatment for ischemic stroke, but apoptosis often occurs in the hypoxic-ischemic environment of the brain after cell transplantation. In this study, we determined if mild hypothermia(27–28°C) can increase the survival rate of neural stem cells(1.0 × 105 /μL) transplanted into neonatal mice with hypoxic-ischemic encephalopathy. Long-term effects on neurological functioning of the mice were also examined. After mild hypothermia combined with neural stem cell transplantation, we observed decreased expression levels of inflammatory factor nuclear factor-kappa B and apoptotic factor caspase-3, reduced cerebral infarct volumes, increased survival rate of transplanted cells, and marked improvements in neurological function. Thus, the neuroprotective effects of mild hypothermia combined with neural stem cell transplantation are superior to those of monotherapy. Moreover, our findings suggest that the neuroprotective effects of mild hypothermia combined with neural stem cell transplantation on hypoxic-ischemic encephalopathy are achieved by anti-inflammatory and anti-apoptotic mechanisms.     

Neural stem cell protects aged rat brain from ischemia–reperfusion injury through neurogenesis and angiogenesis

Neural stem cells (NSCs) show therapeutic potential for ischemia in young-adult animals. However, the effect of aging on NSC therapy is largely unknown. In this work, NSCs were transplanted into aged (24-month-old) and young-adult (3-month-old) rats at 1 day after stroke. Infarct volume and neurobehavioral outcomes were examined. The number of differentiated NSCs was compared in aged and young-adult ischemic rats and angiogenesis and neurogenesis were also determined. We found that aged rats developed larger infarcts than young-adult rats after ischemia (P<0.05). The neurobehavioral outcome was also worse for aged rats comparing with young-adult rats. Brain infarction and neurologic deficits were attenuated after NSC transplantation in both aged and young-adult rats. The number of survived NSCs in aged rats was similar to that of the young-adult rats (P>0.05) and most of them were differentiated into glial fibrillary acidic protein⁺ (GFAP⁺) cells. More importantly, angiogenesis and neurogenesis were greatly enhanced in both aged and young-adult rats after transplantation compared with phosphate-buffered saline (PBS) control (P<0.05), accompanied by increased expression of vascular endothelial growth factor (VEGF). Our results showed that NSC therapy reduced ischemic brain injury, along with increased angiogenesis and neurogenesis in aged rats, suggesting that aging-related microenvironment does not preclude a beneficial response to NSCs transplantation during cerebral ischemia.     

Mutant ubiquitin-mediated ��-secretase stability via activation of caspase-3 is related to ��-amyloid accumulation in ischemic striatum in rats

Previous studies have demonstrated that ischemic stroke increases β-amyloid (Aβ) production by increasing β-secretase (BACE1) through activation of caspase-3, and stimulates generation of mutant ubiquitin (UBB⁺¹) in rat brains. In this study, we examined whether caspase-3 activation participates in the regulation of UBB⁺¹ generation and UBB⁺¹-mediated BACE1 stability in ischemic injured brains. The results showed that UBB⁺¹ and activated caspase-3-immunopositive-stained cells were time dependently increased in the ipsilateral striatum of rat brains after middle cerebral artery occlusion. UBB⁺¹-immunopositive cells could be co-stained with caspase-3, Aβ (UBB⁺¹–Aβ), and BACE1 (UBB⁺¹–BACE1). BACE1 protein could also be pulled down by immunoprecipitation with UBB⁺¹ antibody. Z-DEVD-FMK (DEVD), a caspase-3 inhibitor, significantly decreased the level of UBB⁺¹ protein and the number of UBB⁺¹–Aβ and UBB⁺¹–BACE1 double-stained cells in the ischemic striatum, as well as the level of UBB⁺¹/BACE1 protein complex. We conclude that activation of caspase-3 might be upstream of UBB⁺¹ formation and that excessive UBB⁺¹ could bind to BACE1 and increase the stability of BACE1, thereby increasing Aβ in ischemic injured brains. These results suggest new biological and pathological effects of caspases and regulation of the ubiquitin–proteasome system in the brain. Our results provide new therapeutic targets to prevent further neurodegeneration in patients after stroke.     

Single-cell resolution mapping of neuronal damage in acute focal cerebral ischemia using thallium autometallography

Neuronal damage shortly after onset or after brief episodes of cerebral ischemia has remained difficult to assess with clinical and preclinical imaging techniques as well as with microscopical methods. We here show, in rodent models of middle cerebral artery occlusion (MCAO), that neuronal damage in acute focal cerebral ischemia can be mapped with single-cell resolution using thallium autometallography (TlAMG), a histochemical technique for the detection of the K⁺-probe thallium (Tl⁺) in the brain. We intravenously injected rats and mice with thallium diethyldithiocarbamate (TlDDC), a lipophilic chelate complex that releases Tl⁺ after crossing the blood–brain barrier. We found, within the territories of the affected arteries, areas of markedly reduced neuronal Tl⁺ uptake in all animals at all time points studied ranging from 15 minutes to 24 hours after MCAO. In large lesions at early time points, areas with neuronal and astrocytic Tl⁺ uptake below thresholds of detection were surrounded by putative penumbral zones with preserved but diminished Tl⁺ uptake. At 24 hours, the areas of reduced Tl⁺uptake matched with areas delineated by established markers of neuronal damage. The results suggest the use of ²⁰¹TlDDC for preclinical and clinical single-photon emission computed tomography (SPECT) imaging of hyperacute alterations in brain K⁺ metabolism and prediction of tissue viability in cerebral ischemia.     

In vivo Tracking of Transplanted Bone Marrow-Derived Mesenchymal Stem Cells in a Murine Model of Stroke by Bioluminescence Imaging

Objective

This study was designed to validate the cell trafficking efficiency of the in vivo bioluminescence image (BLI) study in the setting of transplantation of the luciferase expressing bone marrow-derived mesenchymal stem cells (BMSC), which were delivered at each different time after transient middle cerebral artery occlusion (MCAO) in a mouse model.

Methods

Transplanting donor BMSC were prepared by primary cell culture from transgenic mouse expressing luciferase (LUC). Transient focal infarcts were induced in 4-6-week-old male nude mice. The experiment mice were divided into five groups by the time of MSC transplantation : 1) sham-operation group, 2) 2-h group, 3) 1-day group, 4) 3-day group, and 5) 1- week group. BLI for detection of spatial distribution of transplanted MSC was performed by detecting emitted photons. Migration of the transplanted cells to the infarcted area was confirmed by histological examinations. Differences between groups were evaluated by paired t-test.

Results

A focal spot of bioluminescence was observed at the injection site on the next day after transplantation by signal intensity of bioluminescence. After 4 weeks, the mean signal intensities of 2-h, 1-day, 3-day, and 1-week group were 2.6×10⁷ ± 7.4×10⁶, 6.1×10⁶ ± 1.2×10⁶, 1.7×10⁶ ± 4.4×10⁵, and 8.9×10⁶ ± 9.5×10⁵, respectively. The 2-h group showed significantly higher signal intensity (p < 0.01). The engrafted BMSC showed around the infarct border zones on immunohistochemical examination. The counts of LUC-positive cells revealed the highest number in the 2-h group, in agreement with the results of BLI experiments (p < 0.01).

Conclusion

In this study, the results suggested that the transplanted BMSC migrated to the infarct border zone in BLI study and the higher signal intensity of LUC-positive cells seen in 2 hrs after MSC transplantation in MCAO mouse model. In addition, noninvasive imaging in real time is an ideal method for tracking stem cell transplantation. This method can be widely applied to various research fields of cell transplantation therapy.     

Effects of administration route on migration and distribution of neural progenitor cells transplanted into rats with focal cerebral ischemia, an MRI study

We tested the hypotheses that administration routes affect the migration and distribution of grafted neural progenitor cells (NPCs) in the ischemic brain and that the ischemic lesion plays a role in mediating the grafting process. Male Wistar rats (n=41) were subjected to 2-h middle cerebral artery occlusion (MCAo), followed 1 day later by administration of magnetically labeled NPCs. Rats with MCAo were assigned to one of three treatment groups targeted for cell transplantation intra-arterially (IA), intracisternally (IC), or intravenously (IV). MRI measurements consisting of T2-weighted imaging and three-dimensional (3D) gradient echo imaging were performed 24 h after MCAo, 4 h after cell injection, and once a day for 4 days. Prussian blue staining was used to identify the labeled cells, 3D MRI to detect cell migration and distribution, and T2 map to assess lesion volumes. Intra-arterial (IA) administration showed significantly increased migration, a far more diffuse distribution pattern, and a larger number of transplanted NPCs in the target brain than IC or IV administration. However, high mortality with IA delivery (IA: 41% IC: 17% IV: 8%) poses a serious concern for using this route of administration. Animals with smaller lesions at the time of transplantation have fewer grafted cells in the parenchyma.     

Intracerebral transplantation of human adipose tissue stromal cells after middle cerebral artery occlusion in rats

The transplantation of cells capable of neuronal differentiation has great potential for the treatment of neurological conditions. I examined whether human adipose tissue stromal cells (hATSCs) can be induced to undergo neuronal differentiation. I isolated hATSCs from human liposuction tissue and induced neuronal differentiation using azacytidine. After neuronal induction, the hATSCs adopted a more neuronal morphology. These hATSCs were injected into the lateral ventricle of the rat brain, after which they migrated to various parts of the brain. After ischemic brain injury induced by middle cerebral artery occlusion (MCAO), a large number of cells migrated to the injured cortex. Intracerebral grafting of hATSCs significantly enhanced the recovery of functional motor deficits in MCAO rats. These data indicate that transplanted hATSCs survive, migrate and differentiate in the ischemic microenvironment and improve neurological recovery after stroke in rats.     

Inhibition of CXCL12 signaling attenuates the postischemic immune response and improves functional recovery after stroke

After stroke, brain inflammation in the ischemic hemisphere hampers brain tissue reorganization and functional recovery. Housing rats in an enriched environment (EE) dramatically improves recovery of lost neurologic functions after experimental stroke. We show here that rats housed in EE after stroke induced by permanent occlusion of the middle cerebral artery (pMCAO), showed attenuated levels of proinflammatory cytokines in the ischemic core and the surrounding peri-infarct area, including a significant reduction in the stroke-induced chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor-1 (CXCL12). To mimic beneficial effects of EE, we studied the impact of inhibiting CXCL12 action on functional recovery after transient MCAO (tMCAO). Rats treated with the specific CXCL12 receptor antagonist 1-[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclo-tetradecan (AMD3100) showed improved recovery compared with saline-treated rats after tMCAO, without a concomitant reduction in infarct size. This was accompanied by a reduction of infiltrating immune cells in the ischemic hemisphere, particularly cluster of differentiation 3-positive (CD3⁺) and CD3⁺/CD4⁺ T cells. Spleen atrophy and delayed death of splenocytes, induced by tMCAO, was prevented by AMD3100 treatment. We conclude that immoderate excessive activation of the CXCL12 pathway after stroke contributes to depression of neurologic function after stroke and that CXCR4 antagonism is beneficial for the recovery after stroke.     

Endothelial progenitor cell transplantation alleviated ischemic brain injury via inhibiting C3/C3aR pathway in mice

Endothelial progenitor cell transplantation is a potential therapeutic approach in brain ischemia. However, whether the therapeutic effect of endothelial progenitor cells is via affecting complement activation is unknown. We established a mouse focal ischemia model (n = 111) and transplanted endothelial progenitor cells into the peri-infarct region immediately after brain ischemia. Neurological outcomes and brain infarct/atrophy volume were examined after ischemia. Expression of C3, C3aR and pro-inflammatory factors were further examined to explore the role of endothelial progenitor cells in ischemic brain. We found that endothelial progenitor cells improved neurological outcomes and reduced brain infarct/atrophy volume after 1 to 14 days of ischemia compared to the control (p < 0.05). C3 and C3aR expression in the brain was up-regulated at 1 day up to 14 days (p < 0.05). Endothelial progenitor cells reduced astrocyte-derived C3 (p < 0.05) and C3aR expression (p < 0.05) after ischemia. Endothelial progenitor cells also reduced inflammatory response after ischemia (p < 0.05). Endothelial progenitor cell transplantation reduced astrocyte-derived C3 expression in the brain after ischemic stroke, together with decreased C3aR and inflammatory response contributing to neurological function recovery. Our results indicate that modulating complement C3/C3aR pathway is a novel therapeutic target for the ischemic stroke.     

Increased cerebral protein ISGylation after focal ischemia is neuroprotective

Addition of a small peptide called ISG15 is known as ISGylation, which is an ubiquitin (ub)-like posttranslational modification. We currently show that focal ischemia induced by transient middle cerebral artery occlusion (MCAO) in adult mice significantly induces cortical protein ISGylation between 6 and 24 hours reperfusion. With two-dimensional western blotting, 45 proteins were observed to be significantly increased in ISGylation (by 1.8- to 9.7-fold) after focal ischemia compared with sham control. Immunochemistry showed that ISGylated proteins are localized in neurons within the ipsilateral striatum and in astroglia within the peri-infarct cortex of ischemic mice. When subjected to transient MCAO, ISG15^−/− mice showed increased mortality, exacerbated infarction, and worsened neurologic recovery than did wild-type controls. In addition, mice lacking UBE1L (ub-activating enzyme E1-like protein, the first enzyme of the ISGylation cycle) also showed bigger infarcts when subjected to transient MCAO. Regional cerebral blood flow or other physiologic parameters were not significantly different in both knockouts compared with wild-type controls. These studies indicate that increased protein ISGylation might be an endogenous neuroprotective adaptation to minimize poststroke brain damage.     

Autologous bone marrow mononuclear cells enhance recovery after acute ischemic stroke in young and middle-aged rats

We investigated intra-arterially administered autologous bone marrow mononuclear cells (MNCs) in rats with acute ischemic stroke. Long Evans rats (2 to 3 months or 12 months old) underwent tandem reversible common carotid artery (CCA)/middle cerebral artery (MCA) occlusion (CCAo/MCAo) for 3 h and then 24 h later underwent tibial bone marrow harvest. Ten million or 4 million cells were re-injected by an intra-carotid infusion. Control animals underwent marrow needle insertion and then saline injection into the carotid artery. Animals were assessed on a battery of neurological tests. MNCs in the ischemic brain were tracked using Q-dot nanocrystal labeling. Infarct volume and cytokines in the ischemia-affected brain were analyzed. Cell-treated animals in the younger and older groups showed improvement from 7 to 30 days after stroke compared with vehicle-treated animals. MNCs significantly reduced infarct volume compared with saline. There was a significant reduction in tumor necrosis factor-α, interleukin-1α (IL-1α), IL-β, IL-6, and a significant increase in IL-10 in injured brains harvested from the cell-treated groups compared with saline controls. Labeled MNCs were found in the peri-infarcted area at 1 h and exponentially decreased over the ensuing week after injection. Autologous bone marrow MNCs can be safely harvested from rodents after stroke, migrate to the peri-infarct area, enhance recovery, and modulate the post-ischemic inflammatory response.     

脂肪来源干细胞移植对脑缺血大鼠运动功能的影响

目的 观察脂肪来源干细胞(ADAS)移植入脑缺血大鼠后存活、迁移、分化以及大鼠的运动功能障碍恢复情况,探讨ADAS移植治疗大鼠局灶性脑缺血的有效性和可能机制. 方法 将雄性SD大鼠饲养至250～300 g时,制作左侧大脑中动脉阻塞模型(MCAO),按照随机数字表法分为未处理组、对照组和移植组,每组6只.未处理组造模后不作特殊处理,对照组在造模后3h通过尾静脉注射杜氏改良培养基(DMEM),移植组造模后3 h通过尾静脉注射ADAS.造模后14 d处死大鼠,通过免疫荧光染色观察5-溴脱氧尿嘧啶核苷(BrdU)、神经元特异性烯醇化酶(NSE)、人微管相关蛋白2(MAP-2)和神经胶质纤维酸性蛋白(GFAP)的表达.造模后1、7及14 d时神经功能缺损评分评价大鼠运动功能改善情况. 结果 (1)移植后,标记了BrdU的ADAS大量出现在缺血灶周围;(2)MCAO后14d,缺血灶周围出现了少量BrdU/GFAP双染阳性细胞;同时出现少量BrdU/NSE和BrdU/MAP-2双染阳性细胞;(3)14d时移植组大鼠神经功能缺损评分与对照组相比明显降低,差异有统计学意义(P＜0.05). 结论 (1)成年大鼠ADAS在MCAO大鼠体内存活并少量分化为神经元样细胞和星形胶质细胞样细胞;(2)移植ADAS可使大鼠脑缺血所致的运动功能缺损得到改善.