Intracranial bone marrow transplantation after traumatic brain injury improving functional outcome in adult rats

OBJECT: The authors tested the hypothesis that intracranial bone marrow (BM) transplantation after traumatic brain injury (TBI) in rats provides therapeutic benefit. METHODS: Sixty-six adult Wistar rats, weighing 275 to 350 g each, were used for the experiment. Bone marrow prelabeled with bromodeoxyuridine (BrdU) was harvested from tibias and femurs of healthy adult rats. Other animals were subjected to controlled cortical impact, and BM was injected adjacent to the contusion 24 hours after the impact. The animals were killed at 4, 7, 14, or 28 days after transplantation. Motor function was evaluated both before and after the injury by using the rotarod test. After the animals had been killed, brain sections were examined using hemotoxylin and eosin and immunohistochemical staining methods. Histological examination revealed that, after transplantation, BM cells survived, proliferated, and migrated toward the injury site. Some of the BrdU-labeled BM cells were reactive, with astrocytic (glial fibrillary acid protein) and neuronal (NeuN and microtubule-associated protein) markers. Transplanted BM expressed proteins phenotypical of intrinsic brain cells, that is, neurons and astrocytes. A statistically significant improvement in motor function in rats that underwent BM transplantation, compared with control rats, was detected at 14 and 28 days posttransplantation. CONCLUSIONS: On the basis of their findings, the authors assert that BM transplantation improves neurological outcome and that BM cells survive and express nerve cell proteins after TBI.     

Treatment of traumatic brain injury in adult rats with intravenous administration of human bone marrow stromal cells

OBJECTIVE: We investigated the effect of human bone marrow stromal cells (hMSCs) administered intravenously on functional outcome after traumatic brain injury in adult rats. METHODS: hMSCs were harvested from three human donors. A controlled cortical impact was delivered to 27 adult male rats to induce traumatic brain injury, and 24 hours after injury, hMSCs were injected into the tail veins of the rats (n = 18). These rats were divided into two groups: Group 1 was administered 1 x 10(6) hMSCs, and Group 2 was administered 2 x 10(6) hMSCs. Group 3 (control) rats received saline intravenously. Neurological function was evaluated according to the rotarod test and modified neurological severity score. All rats were killed 1 month after injury, and immunohistochemical staining was performed on the brain sections to identify donor hMSCs. To study the phenotypic differentiation of hMSCs, coronal brain sections were stained for neuronal (Tuj1) and astrocytic (glial fibrillary acidic protein) markers. RESULTS: Treatment with 2 x 10(6) hMSCs significantly improved the rats' functional outcomes (P < 0.05). The transplanted cells successfully migrated into injured brain and were preferentially localized around the injury site. Some of the donor cells also expressed the neuronal and astrocytic markers. CONCLUSION: These data suggest that hMSCs may be a potential therapy for patients who have sustained traumatic brain injuries.     

Long-term recovery after bone marrow stromal cell treatment of traumatic brain injury in rats

OBJECT: This study was designed to follow the effects of bone marrow stromal cell (BMSC) administration in rats after traumatic brain injury (TBI) for a 3-month period. METHODS: Forty adult female Wistar rats were injured by a controlled cortical impact and, 1 week later, were injected intravenously with one of three different doses of BMSCs (2 x 10(6), 4 x 10(6), or 8 x 10(6) cells per animal) obtained in male rats. Control rats received phosphate-buffered saline (PBS). Neurological function in these rats was studied using a neurological severity scale (NSS). The rats were killed 3 months after injury, and immunohistochemical stains were applied to brain samples to study the distribution of the BMSCs. Additional brain samples were analyzed by quantitative enzyme-linked immunosorbent assays to measure the expression of the growth factors brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Three months after injury, BMSCs were present in the injured brain and their number was significantly greater in animals that received 4 x 10(6) or 8 x 10(6) BMSCs than in animals that received 2 x 10(6) BMSCs. The cells were primarily distributed around the lesion boundary zone. Functional outcome was significantly better in rats that received 4 x 10(6) or 8 x 10(6) BMSCs, compared with control animals, although no improvement was seen in animals that received 2 x 10(6) BMSCs. All doses of BMSCs significantly increased the expression of BDNF but not that of NGF; however, this increase was significantly larger in animals that received 4 x 10(6) or 8 x 10(6) BMSCs than in controls or animals that received 2 x 10(6) BMSCs. CONCLUSIONS: In summary, when injected in rats after TBI, BMSCs are present in the brain 3 months later and significantly improve functional outcome.     

Treatment of traumatic brain injury in female rats with intravenous administration of bone marrow stromal cells

OBJECTIVE: To study the effect of bone marrow stromal cells administered intravenously to female rats subjected to traumatic brain injury. METHODS: We injected marrow stromal cells harvested from male rat bone marrow (n = 24) into the tail vein of the female rat (n = 8) 24 hours after traumatic brain injury; the rats were killed at Day 7 or 14 after treatment. The neurological function of the rats was evaluated using the rotarod test and the neurological severity score. The distribution of the male donor cells in brain, heart, lung, kidney, liver, muscle, spleen, and bone marrow of the female recipient rats was measured by identifying Y chromosome-positive cells using fluorescent in situ hybridization. RESULTS: We found that marrow stromal cells injected intravenously significantly reduced motor and neurological deficits compared with control groups by Day 15 after traumatic brain injury (P < 0.05, analysis of covariance for repeated measures). The transplanted cells preferentially engrafted into the parenchyma of the injured brain and expressed the neuronal marker NeuN and the astrocytic marker glial fibrillary acidic protein. Marrow stromal cells were also found in other organs in female rats subjected to traumatic brain injury without any obvious adverse effects. CONCLUSION: These data suggest that the intravenous administration of marrow stromal cells may be a promising therapeutic strategy that warrants further investigation for patients with traumatic brain injury.     

Upregulated gene expression of local brain-derived neurotrophic factor and nerve growth factor after intracisternal administration of marrow stromal cells in rats with traumatic brain injury

nourpreviousstudy ,wedemonstratedthatratmarrowstromalcells (rMSCs)migratedintobrainanddifferentiatedintoneuralcellsafterinjectionintoCisternMagnuminadultratssubjectedtotraumaticbraininjury .1Itwasbelievedthatneurologicalfunctionimprovementwaspossiblydueto…     

体外标记神经干细胞脑内移植治疗大鼠创伤性脑损伤

目的 通过不同的方法对神经干细胞（NSCs）进行标记，观察NSCs脑内移植治疗大鼠创伤性脑损伤后的增殖和迁徙情况。方法 Feeney氏法制备大鼠创伤性脑损伤模型，绿色荧光蛋白（GFP）和超顺磁氧化铁（SPIO）标记NSCs后立体定向脑内移植。对移植后大鼠的神经系统行为和运动功能进行评估．免疫组织化学染色观察NSCs的存活及分化情况，并用磁共振成像的方法在体观察NSCs的存活和分布。结果 免疫组织化学检查显示脑内移植后部分GFP阳性细胞表达神经元特异性标记物β-tubilinⅢ，神经系统行为学评分显示移植组动物明显改善．NSCs脑内移植后3周磁共振成像显示移植区低信号改变。结论 移植NSCs可以有效地促进TBI大鼠神经行为功能的恢复，不同的NSCs标记方法能够综合评价细胞移植后的疗效。     

Failure of delayed intravenous administration of bone marrow stromal cells after traumatic brain injury

Traumatic brain injury (TBI) is a leading cause of mortality and morbidity worldwide. Currently, there is no effective strategy to treat the functional sequelae associated with TBI. Experimental evidence shows that the intravenous administration of bone marrow stromal cells (BMSC) during the first week after TBI prevents neurological deficits, but no experimental studies have shown evidence of the effect of intravenous BMSC on chronic brain injury sequelae. Here we studied the effect of intravenous administration of BMSC on functional outcomes 2 months after experimental TBI in rats. Adult Wistar rats were subjected to weight-drop impact causing severe brain injury, and 2 months later BMSC in saline, or saline alone, was intravenously injected. All experimental groups were evaluated by means of the modified Neurological Severity Score (mNSS), and internal zone Permanence Time (izPT) after video-tracking box (VTB) analysis, over the following 2 months to test the efficacy of BMSC therapy. At the end of the study period the animals were sacrificed and their brains were studied to evaluate possible differences between groups. Two months after BMSC administration no significant differences were detected in the motor and sensory evaluation between animals treated with BMSC and controls, and no differences were detected after histological study of the brains. Our present results suggest that intravenous administration of BMSC after TBI, when the neurological deficits are well established, has no beneficial effect in neurological outcomes or on brain tissue.     

Intraarterial administration of marrow stromal cells in a rat model of traumatic brain injury.

To test the efficacy of various delivery routes of stem cells to treat cerebral injury, we investigated the parenchymal distribution of marrow stromal cells (MSCs) injected into the internal carotid artery (ICA) of the adult rat after traumatic brain injury (TBI). Bromodeoxyuridine (BrdU)-labeled MSCs were injected via the ipsilateral ICA at 24 h after TBI. Using histology and immunohistochemistry, the distribution of implanted MSCs was analyzed at 7 days after transplantation. Four groups (n = 4/group) were studied: group 1, animals transplanted with MSCs cultured with NGF and BDNF at 24 h after TBI; group 2, animals transplanted with MSCs cultured without NGF and BDNF; group 3, animals injected with a placebo, phosphate buffered saline into the ICA at 24 h after TBI; and group 4, rats subjected to TBI only. In groups 1 and 2, BrdU-positive cells were localized to the boundary zone of the lesion, corpus callosum and cortex of the ipsilateral hemisphere. The number of BrdU-positive cells was significantly higher in the ipsilateral hemisphere than in the contralateral hemisphere. More MSCs infused intraarterially engrafted in group 1 (18.9%) than in group 2 (14.4%, p < 0.05). Using double staining, BrdU-positive cells expressed MAP-2, NeuN, and GFAP in both groups 1 and 2, with this expression being greater in group 1 and the difference between two groups reaching statistical significance in case of MAP-2. Our data suggest that intraarterial transplantation of MSCs is a viable route for the intracerebral administration of MSCs for the treatment of TBI, since MSCs infused intraarterially after TBI survive and migrate into the brain. Some implanted MSCs express proteins specific to neurons and astrocytes. The addition of NGF and BDNF promote migration of MSCs into the brain and subsequent expression of neuronal protein MAP-2.     

Intravenous administration of marrow stromal cells (MSCs) increases the expression of growth factors in rat brain after traumatic brain injury

This study was designed to investigate the effects of intravenous administration of marrow stromal cells (MSCs) on the expression of growth factors in rat brain after traumatic brain injury (TBI). The fate of transplanted MSCs and expression of growth factors was examined by immunohistochemistry. In addition, the level of growth factors was measured quantitatively using enzyme linked immunosorbent assay (ELISA). Growth factors that were studied included nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF). For immunohistochemical studies, 12 male Wistar rats were subjected to TBI and then divided into three groups with the first group receiving no treatment, the second group receiving saline (placebo) and the third group receiving MSCs intravenously 1 day after TBI. The neurological function of rats was studied by using Rotarod motor test and modified neurological severity scores. The animals were sacrificed 15 days after TBI and brain sections stained by immunohistochemistry to study the distribution of MSCs as well as expression of growth factors NGF, BDNF, and bFGF. For quantitative analysis, a second set of male Wistar rats (n = 18) was subjected to TBI and then injected with either saline (n = 9) or MSCs (n = 9) 1 day after injury. These rats were sacrificed on days 2, 5, and 8 after TBI and brain extracts used to measure NGF, BDNF, and bFGF. We found that after transplantation, MSCs preferentially migrated into the injured hemisphere and there was a statistically significant improvement in the functional outcome of MSC-treated rats compared to control rats. NGF, BDNF, and bFGF were expressed in the injured brain of both treated as well as control rats; however, quantitative ELISA studies showed that expression of NGF and BDNF was significantly increased (p < 0.05) in the treated group. This study shows that intravenous administration of MSCs after TBI increases the expression of growth factors (NGF, BDNF), which possibly contributes to the improvement in functional outcome seen in these rats.     

Combination of bone marrow stromal cell transplantation with mobilization by granulocyte-colony stimulating factor promotes functional recovery after spinal cord transection

Purpose  

Spinal cord injury (SCI) results in severe neurological deficit. However, the functional recovery following SCI is very poor due to the neural lost and limited axonal regeneration. To date, there was no effective treatment. Recent studies have shown that bone marrow stromal cells (BMSCs) transplantated into the central nervous system (CNS) can survive and differentiate into neuronal-like cells. Additionally, granulocyte colony-stimulating factor (G-CSF) can mobilize hematopoietic stem cells and inhibit neural cell apoptosis. Thus, we aimed to evaluate the combined effect of BMSC transplantation and G-CSF administration on rats with traverse spinal cord injury.     

Peripheral nerve regeneration by transplantation of bone marrow stromal cell-derived Schwann cells in adult rats

OBJECT: Bone marrow stromal cells (BMSCs) can be induced to form Schwann cells by sequentially treating the cells with beta-mercaptoethanol and retinoic acid, followed by forskolin and neurotrophic factors including heregulin. In this study the authors made artificial grafts filled with BMSC-derived Schwann cells (BMSC-DSCs) and transplanted them into the transected sciatic nerve in adult rats to evaluate the potential of BMSCs as a novel alternative method of peripheral nerve regeneration. METHODS: The BMSC-DSCs were suspended in Matrigel and transferred into hollow fibers (12 mm in length), which were transplanted into the transected sciatic nerve in adult Wistar rats. Six months after cell transplantation, electrophysiological evaluation and walking track analysis were performed. Results of these studies showed significant improvement in motor nerve conduction velocity and sciatic nerve functional index in the BMSC-DSC-transplanted group compared with the control group (Matrigel graft only). Immunohistochemical study data demonstrated that transplanted BMSCs labeled with retrovirus green fluorescent protein were positive for P0 and myelin-associated glycoprotein and had reconstructed nodes of Ranvier and remyelinated regenerated nerve axons. The number of regenerated axons in the axial section of the central portion of the graft was significantly greater in the transplanted group. Although BMSCs can differentiate into several types of cells, tumor formation did not occur 6 months after engraftment. CONCLUSIONS: Results in this study indicate that BMSC-DSCs have great potential to promote regeneration of peripheral nerves. The artificial graft made with BMSC-DSCs represents an alternative method for the difficult reconstruction of a long distance gap in a peripheral nerve.     

Adenovirus vector-mediated ex vivo gene transfer of brain-derived neurotrophic factor to bone marrow stromal cells promotes axonal regeneration after transplantation in completely transected adult rat spinal cord

The aim of this study was to evaluate the efficacy in adult rat completely transected spinal cord of adenovirus vector-mediated brain-derived neurotrophic factor (BDNF) ex vivo gene transfer to bone marrow stromal cells (BMSC). BMSC were infected with adenovirus vectors carrying β-galactosidase (AxCALacZ) or BDNF (AxCABDNF) genes. The T8 segment of spinal cord was removed and replaced by graft containing Matrigel alone (MG group) or Matrigel and BMSC infected by AxCALacZ (BMSC-LacZ group) or AxCABDNF (BMSC-BDNF group). Axons in the graft were evaluated by immunohistochemistry and functional recovery was assessed with BBB locomotor scale. In the BMSC-BDNF group, the number of fibers positive for growth associated protein-43, tyrosine hydroxylase, and calcitonin gene-related peptide was significantly larger than numbers found for the MG and BMSC-LacZ groups. Rats from BMSC-BDNF and BMSC-LacZ groups showed significant recovery of hind limb function compared with MG rats; however, there was no significant difference between groups in degree of functional recovery. These findings demonstrate that adenovirus vector-mediated ex vivo gene transfer of BDNF enhances the capacity of BMSC to promote axonal regeneration in this completely transected spinal cord model; however, BDNF failed to enhance hind limb functional recovery. Further investigation is needed to establish an optimal combination of cell therapy and neurotrophin gene transfer for cases of spinal cord injury.     

Transplantation of marrow stromal cells restores cerebral blood flow and reduces cerebral atrophy in rats with traumatic brain injury: in vivo MRI study

Cell therapy promotes brain remodeling and improves functional recovery after various central nervous system disorders, including traumatic brain injury (TBI). We tested the hypothesis that treatment of TBI with intravenous administration of human marrow stromal cells (hMSCs) provides therapeutic benefit in modifying hemodynamic and structural abnormalities, which are detectable by in vivo MRI. hMSCs were labeled with superparamagnetic iron oxide (SPIO) nanoparticles. Male Wistar rats (300-350 g, n=18) subjected to controlled cortical impact TBI were intravenously injected with 1 mL of saline (n=9) or hMSCs in suspension (n=9, approximately 3 × 10(6) SPIO-labeled hMSCs) 5 days post-TBI. In vivo MRI measurements consisting of cerebral blood flow (CBF), T2-weighted imaging, and 3D gradient echo imaging were performed for all animals 2 days post-TBI and weekly for 6 weeks. Functional outcome was evaluated with modified neurological severity score and Morris water maze test. Cell engraftment was detected in vivo by 3D MRI and confirmed by double staining. Ventricle and lesion volumetric alterations were measured using T2 maps, and hemodynamic abnormality was tracked by MRI CBF measurements. Our data demonstrate that treatment with hMSCs following TBI diminishes hemodynamic abnormalities by early restoration and preservation of CBF in the brain regions adjacent to and remote from the impact site, and reduces generalized cerebral atrophy, all of which may contribute to the observed improvement of functional outcome.     

Treatment with vitamin B3 improves functional recovery and reduces GFAP expression following traumatic brain injury in rats

Previous studies have shown that administration of vitamin B(3) (B(3)) in animal models of ischemia significantly reduced the size of infarction and improved functional recovery. The present study evaluated the effect of administration of B(3) on recovery of function following traumatic brain injury (TBI), incorporating the bilateral medial frontal cortex contusion injury model. Groups of rats were assigned to B(3) (500 mg/kg) or saline (1.0 ml/kg) treatment conditions and received contusion injuries or sham surgeries. Drug treatment was administered 15 min and 24 h following injury. Rats were examined on a variety of tests to measure sensorimotor performance (bilateral tactile adhesive removal), skilled forelimb use (staircase test), and cognitive ability (reference and working memory) in the Morris Water Maze. Administration of B(3) following injury significantly reduced the behavioral impairments observed on the bilateral tactile removal test, but not on skilled forelimb use. The acquisition of reference and working memory tests were also significantly improved compared to saline-treated rats. Examination of the brains revealed that administration of B(3) significantly reduced the size of the lesion compared to treatment with saline. In addition, examination of glial fibrillary acidic protein (GFAP) expression around the lesion revealed that B(3) significantly reduced the number of GFAP(+) astrocytes. These results indicate that B(3) administration significantly improved behavioral outcome following injury, reduced the size of the lesion, and reduced the expression of GFAP. The current findings suggest that B(3) may have therapeutic potential for the treatment of TBI.     

Delayed intervention with transplants and neurotrophic factors supports recovery of forelimb function after cervical spinal cord injury in adult rats

The adult central nervous system is capable of considerable anatomical reorganization and functional recovery after injury. Functional outcomes, however, vary greatly, depending upon size and location of injury, type and timing of intervention, and type of recovery and plasticity evaluated. The present study was undertaken to assess the recovery of skilled and unskilled forelimb function in adult rats after a C5/C6 spinal cord over-hemisection and delayed intervention with fetal spinal cord transplants and neurotrophins. Recovery of forelimb function was evaluated during both target reaching (a skilled behavior) and vertical exploration (an unskilled behavior). Anatomical tracing and immunohistochemistry were used to assess the growth of descending raphespinal, corticospinal, and rubrospinal fibers at the injury site, tracts that normally confer forelimb function. Delayed intervention with transplants and either brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) restored skilled left forelimb reaching to pre-injury levels. Animals showed recovery of normal reaching movements rather than compensation with abnormal movements. Transplants and NT-3 also improved right forelimb use during an unskilled vertical exploration, but not skilled right reaching. Intervention with fetal transplant tissue supported the growth of descending serotonergic, corticospinal, and rubrospinal fibers into the transplant at the lesion site. The addition of neurotrophins, however, did not significantly increase axonal growth at the lesion site. These studies suggest that the recovery of skilled and unskilled forelimb use is possible after a large cervical spinal cord injury following delayed intervention with fetal spinal cord and neurotrophins. Plasticity of both spared and axotomized descending pathways likely contributes to the functional recovery observed.