Proliferative potential of vascular components in human glioblastoma multiforme

Summary Sixteen patients with glioblastoma multiforme received a 1-h intravenous infusion of 5-bromodeoxyuridine (BrdU), 150–200 mg/m² at the start of surgery, to label S-phase cells in tumor tissue. Labeled cells of vascular components and of tumor parenchyma were detected in excised tumor specimens by indirect immunoperoxidase staining using anti-BrdU monoclonal antibodies followed by periodic acid-Schiff staining. The BrdU labeling index (LI, defined as the percentage of labeled cells in relation to the total number of cells scored) was calculated separately for vascular components and tumor parenchyma in each specimen. The BrdU LI of vascular components of glioblastoma multiforme was remarkably higher than that of normal brain (1.1%–8.7% vs.<0.05%). The mean BrdU LIs of vascular components and tumor cells in eight primary glioblastomas were 4.5±0.8% (mean±SE) and 9.9±1.1%, respectively, while the corresponding BrdU LIs in eight recurrent tumors were 2.7±0.5% and 9.3±0.7%. The differences in the BrdU LIs of primary and recurrent tumors were not statistically significant, but the BrdU LI of vascular components was consistently much lower than that of tumor cells. BrdU labeling of vascular components was inconsistent and occurred mostly in glomerular-shaped vessels, but only about 20% of them contained labeled cells. These results suggest that unusual vascular proliferation, such as the formation of glomerular-shaped vessels and endothelial or adventitial proliferation, in glioblastoma multiforme may have been programmed to slow down or even to cease at a certain stage, and is not likely to be the result of neoplastic transformation.Supported in part by grant PDT-159 from the American Cancer Society and by grant CA 13525 from the National Cancer Institute     

Rapid detection of S-phase cells by anti-bromodeoxyuridine monoclonal antibody in 9L brain tumor cells in vitro and in situ

Summary FITC-conjugated anti-bromodeoxyuridine (BrdUrd) monoclonal antibody (anti-BU-MAb) was used to detect S-phase cells of 9L rat brain tumor cells in vitro and in situ. Monolayer 9L cells were treated with 0.625–20 M of BrdUrd for 30 min, harvested, and reacted with a 1:100 dilution of FITC-conjugated anti-BU-MAb and analyzed with a flow cytometer. BrdUrd-treated cells stained satisfactorily with antibody. Values obtained for the labeling index using this method (48.6%) were 10%–20% higher than the fraction of cells in S-phase calculated from DNA histograms or as the labeling index calculated from autoradiographs of cells pulse-labeled with³H-thymidine. BrdUrd (1–40 mg/kg) was administered by i.p. injection to rats bearing 9L brain tumors. Single cell suspensions obtained by disaggregation of excised tumors were stained with anti-BU-MAb. The percent-age of fluorescent cells (15.9%) calculated using this method was similar to that of S-phase cells (17.2%) calculated from DNA histograms and from autoradiographics for tumor bearing rats pulse-labeled with³H-thymidine in situ. The antibody staining technique is a rapid and accurate method for various cell kinetic studies both in vitro and in vivo in a rat model, and has promise as a technique for the study of cell kinetics in humans.Supported in part by grants PDT159 from the American Cancer Society, CA-13525 from National Cancer Institute and the gift from Aaron Silvera Cancer Research Fund     

Radiohistochemical localization of insulin receptors in the adult and developing rat brain

The light microscopic localization of insulin receptors in the rat central nervous system has been investigated by means of a new autoradiographic technique. These receptors are fairly evenly distributed in the adult brain except in the olfactory bulb. There, the external plexiform layer is especially rich in them. Insulin binding sites were more heterogeneously distributed in the fetal brain and exhibited a waxing and waning during development. Highest densities were observed at 15 days of gestation.     

骨髓间充质干细胞向脑胶质瘤趋向性的初步研究

背景骨髓间充质干细胞(MSCs)是存在于骨髓中的非造血类干细胞,具有自我更新及多向分化潜能.在大鼠脑外伤模型中的迁移能力已得到证实,而其对于脑胶质瘤的趋向性的研究尚处于初期.本实验通过将标记的MSCs移植到大鼠脑胶质瘤模型体内,观察它的迁移情况.方法采用全骨髓细胞培养法,利用MSCs的贴壁生长及可在体外长期培养的特性,获取纯化的MSCs.采用流式细胞术鉴定其表面抗原及细胞周期.在处于对数生长期的MSCs培养皿中加入BrdUrd(终浓度10μg/mL),培养24～48 h后进行移植.采用立体定向技术建立大鼠脑胶质瘤模型,3 d后移植标记好的MSCs在大脑半球组,BrdUrd标记的MSCs被注入大鼠脑胶质瘤模型肿瘤对侧大脑;在颈内动脉组,BrdUrd标记的MSCs被注入大鼠脑胶质瘤模型肿瘤同侧的颈内动脉.分别以BrdUrd标记的3T3细胞作为对照组.2周后,处死大鼠取脑组织制作病理切片,进行抗BrdUrd免疫组化及免疫荧光染色,观察MSCs的迁移情况.结果全骨髓培养法获得了纯化的MSCs.移植到脑组织及颈内动脉的BrdUrd标记的MSCs表现出了明显的向脑胶质瘤迁移的特性.在大脑半球组,MSCs主要集中在肿瘤与正常脑组织的交界部位,在瘤内只有少量分布.在颈内动脉组,MSCs主要分布于肿瘤内部,在肿瘤与正常脑组织的交界位置有少量分布.结论MSCs具有明显的向脑胶质瘤迁移的特性,同时亦可通过血脑屏障,有可能成为胶质瘤基因治疗的理想载体.     

Regional blood flow of experimental brain tumors with special reference to effects of chemotherapy and radiotherapy

Regional blood flow and capillary permeability in the experimental brain tumors and their surrounding brain tissue of rats were measured with quantitative 14C-antipyrine and 14C-alpha-aminoisobutyric acid (AIB) autoradiographic method. The pharmacokinetic implications with respect to drug delivery to tumor tissue and the effect of ionizing irradiation were discussed in these physiological measurement. A suspension of 1 X 10(4) rat glioma cells (E239 RG 12) was stereotactically implanted into the right basal ganglia of CD-Fisher rats, and spherical brain tumors developed 10-17 days after implantation with a diameter of 1--5 mm. Autoradiographic investigations were performed for rats with small tumors (1--2 mm in diameter) and large tumors (4--5 mm in diameter). The uniform blood flow (91.7 +/- 13.1 ml/100 g/min: mean +/- S.E.) was observed in small tumors with the patchy low flow area (56.7 +/- 12.5 ml/100 g/min) surrounding the tumor. In large tumors, the blood flow was markedly decreased in the central part of the tumor (28.3 +/- 2.4 ml/100 g/min) with a ring shaped high flow area in the peripheral part (59.3 +/- 5.9 ml/100 mg/min). The blood flow in the brain adjacent to the tumor (30.5 +/- 2.5 ml/100 g/min) was lower than that in the peripheral part of the tumor. The uptake of 14C-AIB was quite similar to that of 14C-antipyrine suggesting the smaller permeability in the central part of the tumor. Neuropathological studies did not reveal necrotic foci, but viable cells in these areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical detection of cells labeled with bromodeoxyuridine after neural transplantation

Pregnant rats were treated with 5-bromo-2′-deoxyuridine (BrdU) from embryonic d 12 (ED12) to ED14. BrdU administration allowed timed labeling of dividing embryonic cellsin utero, since the drug is incorporated into the DNA in place of thymidine during the S-phase of the cell cycle. ED14 rat cerebral cortex or placenta was grafted into the brain of adult rats. Anti-bromodeoxyuridine immunohistochemistry was used for identifying labeled transplanted cells after different survival periods in paraffin-embedded sections. BrdU labeled cells were observed in both intraventricular and intraparenchymal cortical grafts, even after a 3-mo survival. Although the percentage of positive cells decreased in comparison with ED14 cortex, the level of BrdU (i.e., the intensity of anti-BrdU immunohistochemistry) in labeled nuclei was probably the same. BrdU pretreatment of ED14 cells prior to grafting did not affect the proliferative ability of the grafted tissue. In ED14 placental grafts, all trophoblastic cells were labeled distinctly. This precise labeling technique enabled an examination of individual migrating trophoblastic cells. However, migration of these cells into the host brain was very limited.     

Selective enhancement of tumor blood flow and drug delivery to brain tumors in experimental rat gliomas under angiotensin II-induced hypertension

Regional blood flow of brain tumors and normal brain tissue of rats before and during angiotensin II (AT II)-induced hypertension were measured using an electrolytic flowmeter and a laser flowmeter. Etoposide concentration in the tumor and brain tissue after intracarotid administration were also measured in brain tumor bearing rats with or without AT II-induced hypertension. A suspension of 5 x 10(5)/10 microliters 9L gliosarcoma cells was inoculated into the left caudate-putamen of CD Fischer 344 rats. Before induced hypertension, regional blood flow of the tumor (28.2 +/- 2.6 ml/100 g/min; mean +/- SEM) and the contralateral caudate-putamen (23.0 +/- 1.8 ml/100g/min) in the tumor bearing rats were significantly lower than that of the caudate-putamen (43.9 +/- 4.1 ml/100g/min) in the normal rats (p less than 0.01). Intravenous administration of AT II at a dose of 0.4-0.6 microgram/body/min increased the mean arterial blood pressure from 96.5 +/- 4.7 mmHg to 138.0 +/- 3.6 mmHg. AT II-induced hypertension resulted in an approximate 1.8(1.1 - 3.6)-fold increase in the regional tumor blood flow. On the other hand the regional blood flow of the contralateral caudate-putamen was slightly decreased at the rate of 6%. The mean concentration of etoposide with AT II-induced hypertension in the tumor tissue was 2.2-fold higher than that without AT II-induced hypertension. However, etoposide delivery to normal brain tissue was small. From these results, induced hypertension with intravenously administrated AT II selectively increase the tumor blood flow and drug delivery to brain tumor tissue. Intracarotid chemotherapy with AT II-induced hypertension might contribute to enhance therapeutic effect of malignant brain tumors.     

Autoradiographic localization of peripheral benzodiazepine binding sites in the cat brain with [3H]PK 11195

"Peripheral type" benzodiazepine binding sites were labelled in cat brain membranes by using [3H]PK 11195. This ligand binds to the "peripheral type" binding sites in a reversible, specific and saturable manner. Cat brain binding sites density (congruent to 6 pmol/mg prot.) was higher than in the rat. Pharmacological specificity was demonstrated by the potency order of displacing agents: PK 11195 greater than RO5-4864 greater than dipyridamole greater than diazepam greater than clonazepam. A similar characterization was performed in slide mounted brain sections where [3H]PK 11195 also labelled the "peripheral type" benzodiazepine binding sites. The high percentage of specific binding (80%) at 1 nM of [3H]PK 11195 made possible the autoradiographic studies on binding sites distribution. These sites were heterogeneously distributed in the grey matter and absent in white matter. Visual, auditory and other specific sensory relay stations were highly labelled. The blood pressure regulating nuclei, the vestibulo-cerebellar and the extrapyramidal motor system also presented a very high binding density. As previously described in the rat brain, choroid plexus was also strongly labelled by [3H]PK 11195 in the cat.     

A glial fibrillary acidic protein-expressing and tumorigenic cell line derived from an avian sarcoma virus-induced rat astrocytoma

A permanent cell line, S635c15, was derived from an anaplastic astrocytoma induced by the Schmidt-Ruppin strain of avian sarcoma virus (ASV) in a female F-344 rat. Persistent expression of the astrocytic differentiation protein, glial fibrillary acidic protein (GFAP), was detected both in cultured cells after 100 passages in vitro and in transplanted tumors. Subcutaneous and intracerebral transplantation of S635c15 cells in syngeneic rats resulted in a 100% tumor incidence and a reproducible mortality distribution. S635c15 cells formed discrete masses after subcutaneous injection but grew intracranially as infiltrative lesions. Tumor blood flow and blood-to-tissue transport studies yield comparable values to other rat glioma models; S635c15 intracranial tumors proved to be a homogeneous model with little variation within and between tumors with respect to morphology, GFAP expression, blood flow, and permeability. This cell line provides a GFAP-expressing brain tumor model that extends the use of autochthonous ASV-induced astrocytomas by allowing in vitro and in vivo studies. It may be useful for further studies in neurobiology and brain tumor biology, diagnosis, and therapy.     

Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus.

Knowing the rate of addition of new granule cells to the adult dentate gyrus is critical to understanding the function of adult neurogenesis. Despite the large number of studies of neurogenesis in the adult dentate gyrus, basic questions about the magnitude of this phenomenon have never been addressed. The S-phase marker bromodeoxyuridine (BrdU) has been extensively used in recent studies of adult neurogenesis, but it has been carefully tested only in the embryonic brain. Here, we show that a high dose of BrdU (300 mg/kg) is a specific, quantitative, and nontoxic marker of dividing cells in the adult rat dentate gyrus, whereas lower doses label only a fraction of the S-phase cells. By using this high dose of BrdU along with a second S-phase marker, [(3)H]thymidine, we found that young adult rats have 9,400 dividing cells proliferating with a cell cycle time of 25 hours, which would generate 9,000 new cells each day, or more than 250,000 per month. Within 5-12 days of BrdU injection, a substantial pool of immature granule neurons, 50% of all BrdU-labeled cells in the dentate gyrus, could be identified with neuron-specific antibodies TuJ1 and TUC-4. This number of new granule neurons generated each month is 6% of the total size of the granule cell population and 30-60% of the size of the afferent and efferent populations (West et al. [1991] Anat Rec 231:482-497; Mulders et al. [1997] J Comp Neurol 385:83-94). The large number of the adult-generated granule cells supports the idea that these new neurons play an important role in hippocampal function.     

Cell kinetic analysis of human brain tumors by bivariate flow cytometric measurement of cellular DNA content and amount of incorporated bromodeoxyuridine

Cell kinetics of 91 human brain tumors obtained from 88 patients were analyzed with the following two methods, 1) bivariate (two-color) flow cytometric measurement of cellular DNA content and amount of bromodeoxyuridine (BrdU) incorporated into cellular DNA, in 66 specimens, 2) immunohistochemical detection of BrdU incorporated S-phase cells, in 34 specimens. Patients were given an intravenous 1 hour infusion of 200 mg/sq. m. of BrdU 1-2 hours before the surgical removal. The excised tumor specimen was divided into several portions. One was fixed with 70% ethanol and embedded in paraffin, and another was digested mechanically and/or chemically to obtain a single cell suspension, and fixed in 70% ethanol. Paraffin-embedded tissue sections were stained by the peroxidase-antiperoxidase immunohistochemical method using anti-BrdU monoclonal antibody (MoAb). Single cell suspensions were reacted with fluorescein isothiocyanate (FITC) conjugated anti-BrdU MoAb, or anti-BrdU MoAb and FITC-conjugated second antibody successively by the staining with propidium iodide, for flow cytometry (FCM). Rates of S-phase fraction in single cell suspensions calculated by bivariate FCM were correlated well with labeling indexes (LI, i.e. the percentage of BrdU incorporated cells) calculated in tissue sections, but not with the result of analysis of DNA histogram by Dean's method. This discrepancy is probably due to large coefficient value in several samples. Histological malignancy of the tumors was reflected both in the proliferating index (PI, i.e. % S+G2M phase) calculated by bivariate FCM and the LI by immunohistochemical method. PI tended to be high in primitive neuroectodermal tumors and metastatic carcinomas, moderately high in gliomas, and low in benign tumor groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local cerebral blood flow and glucose metabolism in experimental meningeal carcinomatosis

Patients with meningeal carcinomatosis often evolve signs of impairment in higher mental function. Yet, common findings of histological observation are only a sheet of tumor cells on the cortical surface, and no intracerebral mass are noted. To elucidate mechanism of mental disturbances in meningeal carcinomatosis, local cerebral blood flow and glucose metabolism were evaluated in a model of experimental meningeal carcinomatosis. Viable cells (1 X 10(4) of Walker 256 tumor were inoculated into cisterna magna of Wistar rats. Animals were used for autoradiographic study at 1 to 12 days after tumor inoculation. Local cerebral glucose utilization (LCGU) and local cerebral blood flow (LCBF) were measured with quantitative autoradiographic technique using 14C-iodoantipyrine and 14C-deoxyglucose as a tracer, respectively. In the early stage of tumor growth (1 to 3 days after tumor inoculation), reduction of LCGU was averaged to be 31% in the cerebral cortex and 28% in the deep structures, whereas reduction of LCBF was 28% in cerebral cortex and 19% in deep structures on average. In the late stage of tumor growth (4 to 12 days after tumor inoculation), average reduction of LCGU was 57% in the cerebral cortex and 47% in the deep structures. On the other hand, reduction of LCBF was averaged to be 42% in the cerebral cortex and 38% in the deep structures in the late stage of the disease. Reduction of LCGU and LCBF was especially evident in the sensory cortices such as parietal cortex, visual cortex and auditory cortex, and in the auditory centers of the brain stem such as medial geniculate body and inferior colliculus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Postnatal changes in local cerebral blood flow measured by the quantitative autoradiographic [14C]iodoantipyrine technique in freely moving rats

The postnatal changes in local cerebral blood flow in freely moving rats were measured by means of the quantitative autoradiographic [14C]iodoantipyrine method. The animals were studied at 10, 14, 17, 21 and 35 days and at the adult stage. At 10 days after birth, rates of blood flow were very low and quite homogeneous in most cerebral structures except in a few posterior areas. From these relatively uniform levels, values of local cerebral blood flow rose notably to reach a peak at 17 days in all brain regions studied. Rates of blood flow decreased between 17 and 21 days after birth and then increased from weaning time to reach the known characteristic distribution of the adult rat. The postnatal evolution of local cerebral blood in the rat is in good agreement with previous studies in other species such as dog and humans that also show higher rates of cerebral blood flow and glucose utilization at immature stages. However, in the rat, local cerebral blood flow and local cerebral glucose utilization are not coupled over the whole postnatal period studied, since blood flow rates reach peak values at 17 days whereas glucose utilization remains still quite low at that stage. The high rate of cerebral blood flow in the 17-day-old rat may reflect the energetic and biosynthetic needs of the actively developing brain that are completed by the summation of glucose and ketone body utilization.     

The effects of the GABAergic agonist muscimol upon the relationship between local cerebral blood flow and glucose utilization

The relationship between cerebral blood flow and glucose utilization was studied in conscious rats using the [14C]iodoantipyrine and [14C]2-deoxyglucose quantitative autoradiographic techniques respectively. Both blood flow and glucose use decreased in parallel in the 38 areas of the brain analyzed following administration of the GABA agonist muscimol, thus maintaining the relationship observed in control rats. The present study offers no evidence for a direct vasodilatatory action for muscimol in the conscious rat, but points to underlying regional metabolic activity as the primary determinant of cerebral blood flow.     

Use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system

The use of an exogenously administered thymidine analog, 5-bromo-2'-deoxyuridine (BrdU), for studies of the proliferation, migration and time of origin of cells in the cerebral cortex was investigated and compared with [3H]thymidine [( 3H]dT) autoradiography. Pregnant rats or mice were injected with BrdU and/or [3H]dT and processed by standard immunohistochemical techniques using a primary antibody directed against BrdU in single-stranded DNA, autoradiographic methods, or both. In animals that survived only 1 h after the injection, BrdU-positive cells were distributed in the proliferative zones throughout the central nervous system (CNS). In animals killed 1-3 days after the BrdU injection, intensely immunoreactive cells were in the superficial cortical plate and less intensely labeled cells were scattered throughout the deep cortical plate, the intermediate zone, and the germinal zones. In adult animals, 60 days or more after an injection of BrdU on GD 19, BrdU-positive cells were located in layer II/III of neocortex, the hippocampal pyramidal layer, and the granule layer of the dentate gyrus. In the double-labeling studies, the distribution of BrdU-immunoreactive cells was identical to that of autoradiographically labeled cells, and all autoradiographically labeled neurons were BrdU positive. Thus, BrdU immunohistochemistry is suitable for developmental studies of the CNS; moreover, it provides several advantages over [3H]dT autoradiography.     

Postnatal cell proliferation in the rat cerebrum: immunohistochemical study with bromodeoxyuridine (BrdU)

The postnatal cell proliferation in the rat cerebrum was studied immunohistochemically using a monoclonal antibody to bromodeoxyuridine (BrdU). Since BrdU, a halogenated analogue of thymidine, is incorporated into nuclear DNA during duplication, S-phase cells can be detected by demonstrating intranuclear BrdU. 200 mg/kg of BrdU was administered to normal Wistar rats intraperitoneally on the day of birth or intravenously 1, 2, 3, 5, 8 or 24 weeks after birth. Thirty minutes later, the brain was fixed by perfusion with ethanol, and the paraffin-embedded sections were processed for the avidin biotin peroxidase-complex method. BrdU-positive nuclei were counted among 500 to 10,000 cells in several regions of the brain to obtain the BrdU-labeling index (the number of BrdU-positive cells per 100 cells scored, LI, %). The present study demonstrated that (1) proliferating cells in the gray matter (cerebral cortex and caudate-putamen) are only few at birth (LI = 0.54-0.78%), which further decrease during the following few weeks, and disappear by adulthood, (2) in the white matter (corpus callosum), cell proliferation is relatively active within 1 week after birth (LI = 5.6-6.3%), but becomes inactive thereafter, (3) the proliferative activity of the cells in the subependymal layer of the lateral ventricle is very high at birth (LI = 15.5%), which somewhat decreases during the following few weeks, but still remains high in adulthood (LI = 7.5%). This kind of continued cell proliferation in the brain after birth seems important in the postnatal development of the normal cerebral structure, and in several pathologic processes such as tissue repair and the development of brain neoplasm.     

Cortical neurogenesis in adult rats after reversible photothrombotic stroke.

Neurogenesis occurs throughout life in the dentate gyrus of hippocampus and subventricular zone, but this phenomenon has rarely been observed in other brain regions of adult mammals. The aim of the current study was to investigate the cell proliferation process in the ischemically challenged region-at-risk after focal cerebral ischemia in the adult rat brain. A reversible photothrombotic ring stroke model was used, which features sustained hypoperfusion followed by late spontaneous reperfusion and a remarkable morphologic tissue recovery in the anatomically well defined somatosensory cortical region-at-risk. Twelve-week-old male Wistar rats received repeated intraperitoneal injections of the cell proliferation specific marker 5-bromodeoxyuridine (BrdU) after stroke induction. Immunocytochemistry of coronal brain sections revealed that the majority of BrdU-positive cells were of glial, macrophage, and endothelial origin, whereas 3% to 6% of the BrdU-positive cells were double-labeled by BrdU and the neuronspecific marker Map-2 at 7 and 100 days after stroke onset in the region-at-risk. They were distributed randomly in cortical layers II-VI. Three-dimensional confocal analyses of BrdU and the neuronal-specific marker Neu N by double immunofluorescence confirmed their colocalization within the same cells at 72 hours and 30 days after stroke induction. This study suggests that, as a potential pathway for brain repair, new neurons can be generated in the cerebral cortex of adult rats after sublethal focal cerebral ischemia.     

Distribution and differentiation of bone marrow-derived mesenchymal stem cells in vivo after intraperitoneal and tail vein injection into rats in the recovery phase of stroke Which path is better?

BACKGROUND:Stereotactic injection(striatum or lateral ventricle)and vascular injection(tail vein or carotid artery)are now often used in cellular therapy for cerebral infarction.Stereotactic injection can accurately deliver cells to the infarct area,but requires a stereotactic device and causes secondary trauma;vascular injection is easy and better for host neurological deficit recovery,but can cause thrombosis.OBJECTIVE: To compare the therapeutic potential of adult bone marrow-derived mesenchymal stem cells(BMSCs)transplantation by intraperitoneal versus intravenous administration to cerebral ischemic rats.DESIGN,TIME AND SETTING: A randomized controlled animal experiment was performed at the Cell Room and Pathology Laboratory,Brain Hospital Affiliated to Nanjing Medical University from November 2007 to September 2008.MATERIALS: BMSCs were derived from 20 healthy Sprague-Dawley rats aged 4-6 weeks.METHODS: Forty-five adult middle cerebral artery occlusion(MCAO)rats were randomly divided into control,intravenous and intraperitoneal injection groups,with 15 rats in each group.At 21 bromodeoxyuridine(BrdU)via intravenous or intraperitoneal injection.MAIN OUTCOME MEASURES: Angiogenin expression and survival of transplanted cells were measured by immunohistochemical staining of brain tissue in infarction hemisphere at 7,14 or 21 days after BMSC transplantation.Co-expression of BrdU/microtubule-associated protein 2 or BrdU/glial fibrillary acidic protein was observed by double-labeled immunofluorescence of cerebral cortex.Evaluation of nerve function using the neurological injury severity score and the adhesion-removal test was performed on the 1st and 21st day before and after MCAO,and at 3,7,14 or 21 days after BMSCs treatment.RESULTS: Angiogenin-positive new vessels were distributed in the bilateral striatum,hippocampus and cerebral cortex of each group of rats at each time point,most markedly in the intravenous injection group.There were significantly more BrdU-positive cells in the intravenous injection group than in the intraperitoneal injection group(P < 0.01).Co-expression of BrdU/ microtubule-associated protein 2 or BrdU/glial fibrillary acidic protein were almost only seen in theintravenous group by fluorescence microscopy.After transplantation,BMSCs significantly restored nerve function in rats,particularly in the intravenous injection group.CONCLUSION: BMSCs were able to enter brain tissue via the tail vein or peritoneal injection and improve neurological function by promoting the regeneration of nerves and blood vessels in vivo,more effectively after intravenous than intraperitoneal injection.     

The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis

Adult animals continue to produce new neurons in the dentate gyrus of hippocampus. Until now, the principal method of studying neurogenesis has been to inject either tritiated thymidine or 5'-Bromo-2-deoxyuridine (BrdU) intraperitoneally followed by autoradiographic or immunohistochemical detection methods respectively. However, such exogenous markers may produce toxic effects. Our objective was to determine whether Ki-67, a nuclear protein expressed in all phases of the cell cycle except the resting phase, can be used as an alternative, endogenous marker. Using immunohistochemistry, we examined Ki-67 and BrdU expression pattern in rats. Ki-67 was expressed within the proliferative zone of the dentate gyrus and its expression pattern mimicked that of BrdU when examined soon after exogenous BrdU administration. Quantitative comparison of BrdU and Ki-67-positive cells showed 50% higher numbers of the latter when examined 24 h after the BrdU injection. This was expected, since BrdU can be incorporated into DNA only during the S-phase of the mitotic process, whereas Ki-67 is expressed for its whole duration. Experimental increases (by ischemia) or reductions (by radiation) in the number of mitotic cells produced parallel changes in BrdU and Ki-67 signals. Thus, Ki-67 is an effective mitotic marker and has most of the benefits of BrdU and none of the costs. This study provides evidence for Ki-67 to be used as a marker of proliferation in the initial phase of adult neurogenesis.