Intravenous administration of bone marrow mononuclear cells alleviates hearing loss after transient cochlear ischemia through paracrine effects.

Degeneration and repair 807

Intravenous administration of bone marrow mononuclear cells alleviates hearing loss after transient cochlear ischemia through paracrine effects Taro Takagi, Tadashi Yoshida, Masahiro Okada, Ryuji Hata, Naohito Hato, Kiyofumi Gyo and Nobuhiro Hakuba Bone marrow mononuclear cells (BMMCs) are known to enhance recovery from ischemic insults by secreting angiogenic factors and inducing the expression of angiogenic factors from host tissues. Therefore, the transplantation of BMMCs is considered a potential approach to promoting the repair of ischemic damaged organs. Here, we investigated the influence of BMMCs on progressive hair cell degeneration after transient cochlear ischemia in gerbils. Transient cochlear ischemia was produced by extracranial occlusion of the bilateral vertebral arteries immediately before their entry into the transverse foramen of the cervical vertebra. An intravenous injection of BMMCs prevented ischemia-induced hair cell degeneration and ameliorated hearing impairment. A tracking study showed that BMMCs injected into the femoral vein were limited in the spiral artery of the cochlea, suggesting that, although transplanted BMMCs were retained within the spiral ganglion area of the cochlea, they were neither transdifferentiated into cochlear cells nor fused with the injured hair cells and supporting

cells in the organ of Corti to restore their functions. We also showed that the protein level of neurotrophin-3 and glial cell line-derived neurotrophic factor in the organ of Corti was upregulated after treatment with BMMCs. These results suggested that BMMCs have therapeutic potential possibly through paracrine effects. Thus, we propose the use of BMMCs as a potential new therapeutic strategy for c 2014 Wolters hearing loss. NeuroReport 25:807–813 Kluwer Health | Lippincott Williams & Wilkins.

Introduction

completed without ex-vivo culture, making BMMCs the most suitable candidate cells for therapeutic neovascularization.

Most experimental investigations on cell-based therapeutic angiogenesis have achieved satisfactory alterations in ischemic hearts and brain dysfunction [1,2]. Clinical trials have also shown the safety and feasibility of implanting autologous peripheral blood or bone marrow-derived cells (BMMCs) for the treatment of ischemic heart disease, brain disease, and peripheral arterial disease by increasing regional blood flow, with improvement in clinical data [3–5]. BMMCs, including endothelial progenitor cells (EPCs) and marrow stromal cells (MSCs), are known to induce neovascularization in ischemic tissues, leading to enhanced recovery from ischemic insults [6,7]. EPCs increase neovascularization by transdifferentiating into endothelial cells, secreting angiogenic factors, and inducing the expression of angiogenic factors from host tissues [8,9]. MSCs express a broad spectrum of angiogenic factors. The local injection of MSCs is also reported to increase the levels of angiogenic factors in vivo and in vitro, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) [7]. However, the culture of EPCs and MSCs is time consuming and is technically difficult for clinical applications. By contrast, isolation of BMMCs could be c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4965

NeuroReport 2014, 25:807–813 Keywords: bone marrow mononuclear cell, cell therapy, cochlear ischemia, glial cell line-derived neurotrophic, hair cell death, hearing loss, neurotrophin-3 Ehime University School of Medicine, Toon, Ehime, Japan Correspondence to Nobuhiro Hakuba, MD, PhD, Ehime University School of Medicine, Shizugawa-cho, Toon, Ehime 791-0295, Japan Tel: + 81 89 960 5368; fax: + 81 89 960 5366; e-mail: [email protected] Received 21 February 2014 accepted 13 March 2014

However, idiopathic sudden sensorineural hearing loss is an acute form of hearing loss, and its etiologies remain controversial. Although various etiologies have been proposed, acute interruption of the blood supply to the cochlea is considered one of the major causes of sudden deafness [10]. We reported previously the effects of cell-based therapy on a transient cochlear ischemia model [11]. An injection of hematopoietic stem cells (HSCs) through the round window (RW) markedly suppressed hair cell death after transient cochlear ischemia. In the present study, we further explored the feasibility of an intravenous administration of BMMCs as therapy for hearing loss in a transient cochlear ischemia model and documented the homing of donor cells to the injured cochlea because the systemic delivery of stem cells may be considered an alternative option.

Methods The experiments were conducted in accordance with the Guidelines for Animal Experimentation at Ehime University DOI: 10.1097/WNR.0000000000000167

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

808 NeuroReport 2014, Vol 25 No 11

Graduate School of Medicine. The animals received humane care as required by the institutional guidelines and the Guide for the Care and Use of Laboratory Animals [12]. Transient cochlear ischemia and reperfusion

Adult male Mongolian gerbils (Meriones unguiculatus) weighing 60–80 g were used at 8–16 weeks of age. Following the procedures described by Hata et al. [13], transient cochlear ischemia was induced by temporarily occluding bilateral vertebral arteries in the neck because gerbils lack posterior cerebral communicating arteries and the labyrinthine arteries are nourished solely by the vertebral–basilar system. Anesthesia was induced with a mixture of 3% halothane and nitrous oxide: oxygen (7 : 3) gas and maintained with a mixture of 1% halothane gas. The animals were ventilated artificially through a transoral tracheal tube (tidal volume, 1 ml; respiration rate, 70/min). During the experiment, body temperature was monitored using a thermocouple probe (PTI-200; Unique Medical, Tokyo, Japan) placed in the rectum and maintained at 37±11C using a heating plate (HP-1M; Physitemp, Clifton, New Jersey, USA). The vertebral arteries were exposed bilaterally and dissected free from the surrounding tissues through a ventral midline incision in the neck with the animal in the supine position. Silk ligatures (4–0) were looped loosely around each artery. Ischemia was then induced in both cochleae by pulling the ligatures simultaneously using 5-g weights for 15 min. Subsequently, the threads were removed to allow reperfusion, which was confirmed by observation through an operating microscope. The wound was closed and the animals were returned to cages with food and water available ad libitum until further testing. Isolation of BMMCs

Adult male Mongolian gerbils aged 8–16 weeks were used as donors for bone marrow transplantation. Bone marrow cells were obtained from both femoral and tibial bones, and mononuclear cells were isolated by density gradient centrifugation using Ficoll (GE Healthcare, Uppsala, Sweden) at 400g for 40 min according to the manufacturer’s protocol. Administration of BMMCs

To avoid injury to the microvasculature likely to occur with the standard pretreatment for bone marrow transplantation, recipient animals received no radiation or chemotherapy before bone marrow transplantation. Thirty minutes after transient cochlear ischemia, the femoral vein was exposed to establish an intravenous infusion line using a polyethylene catheter. The animals were assigned randomly to receive BMMCs (0.2 ml; 1 106 cells/ml suspended in PBS) (n = 6) or saline (n = 6), induced over 20 min to avoid acute volume load, after which the catheter was removed and the vein was ligated.

Evaluation of hearing by auditory brainstem response

Hearing was assessed before and at 1, 4, and 7 days after ischemia. The animals were anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (1 mg/kg), and then auditory brainstem response (ABR) was recorded using a signal processor (NEC Synax 1200; NEC Medical Systems, Tokyo, Japan). Recording and reference needle electrodes were placed at the vertex and ipsilateral retroauricular area, respectively. As ischemic hearing loss less than 4 kHz was minor in this animal model [14], we used pure tone bursts at 8, 16, and 32 kHz (rise and fall time, 1 ms; duration, 5 ms; repetition rate, 15/s) as auditory stimuli, delivered in an open-field system (DPS-725; DIA Medical, Tokyo, Japan). The phases of the stimuli were alternated, thereby canceling interference of the cochlear microphonics. The speaker was located 20 cm in front of the left external auditory canal and a masking noise (white noise) was administered to the right ear canal through a very small polypropylene tube. Responses to 1000 consecutive stimuli were averaged. The ABR threshold was determined by recording responses in 5-dB increments.

Evaluation of hair cell loss

The animals were killed for histological study 7 days after recording their ABR. Under deep anesthesia, following removal of the otic bullae, the cochleae were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 into the scala tympani and postfixed for 2 h with the same fixative at 41C. The specimens were immersed in PBS and the organ of Corti was dissected using a surface preparation technique under an operating microscope. The walls of the bony cochleae were removed entirely without disrupting the organ of Corti. Next, the basal turn of the organ of Corti was isolated. Each specimen was stained with rhodamine–phalloidin (Molecular Probes, Eugene, Oregon, USA) diluted 250fold in PBS containing 0.25% Triton X-100 and 1% bovine serum albumin for 30 min at room temperature. After rinsing in PBS, the specimen was stained further with Hoechst 33342 (Calbiochem-Novabiochem, La Jolla, California, USA) dissolved in PBS in a dark room for 1 h. The specimen was again rinsed in PBS and mounted in carbonate-buffer glycerol (one part 0.5 M carbonate buffer at pH 9.5 to nine parts glycerol) containing 2.5%1,4-diazabicyclo[2,2,2]octane to retard bleaching of the fluorescent signal. Fluorescence was detected using an Olympus BX60 microscope (Olympus, Tokyo, Japan) equipped with a green band pass (BP) 546, darb teiler spiegel (FT) 580, long pass (LP) 590 nm, and UV (BP 365, FT 395, LP 397 nm) filters. Rhodamine–phalloidin staining enables observation of the hair cell architecture, whereas Hoechst 33342 staining allows visualization of the nuclei. The numbers of intact and dead hair cells at the basal turn were counted and the percentages of dead hair cells and intact hair cells were determined. Gerbils have B300 inner hair cells (IHCs) at the basal turn, and we examined at least 200 IHCs in each specimen.


Intravenous administration of BMMCs Takagi et al. 809

Western blot analysis

After deep anesthesia with an intraperitoneal injection of sodium pentobarbital (0.1 g/kg), the otic bulla (wet weight, 10 mg) was removed and transferred to ice-cold PBS. The samples were homogenized in microcentrifuge tubes containing 100 ml of lysis buffer (0.5% SDS, 0.5% Triton X, 100 mM phenylmethane sulfonyl fluoride, 20 mM Tris–HCl, pH 8.0). The homogenates were sonicated on ice and centrifuged at 13 000 rpm for 10 min at 41C. The protein content in the supernatant was determined using a BCA protein assay kit (Pierce, Rockland, Illinois, USA) with bovine serum albumin as a standard. The supernatant was mixed with sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, and 0.001% bromophenol blue) to a final protein concentration of 1 mg/ml. The samples were then boiled for 5 min. Equal amounts of protein (20 mg/lane) were resolved by SDS-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane, and immunoblotted with an antibody against neurotrophin-3 (NT-3) (sc-547; Santa Cruz Biotechnology, Santa Cruz, California, USA) and glial cell line-derived neurotrophic factor (GDNF) (sc-328; Santa Cruz Biotechnology). Densitometric analysis of the scanned bands was carried out to quantify the NT-3 and GDNF protein levels in the samples. The integrated optical density was obtained using the NIH Image software (National Institutes of Health, Bethesda, Maryland, USA). The data were normalized to internal standards (PBS-treated control) on each gel and expressed as percentages.

were viewed using an Olympus BX60 fluorescence microscope. Statistical analysis

All data are presented as the means±SD. The significance of differences between the groups was evaluated using the Mann–Whitney U-test. The results were considered to be statistically significant at a P value less than 0.05.

Results Hearing loss

Figure 1 shows the sequential changes in the ABR threshold at 8, 16, and 32 kHz, before and 1, 4, and 7 days after the induction of transient cochlear ischemia. The preischemic ABR threshold was defined as 0 dB and the subsequent increase in threshold was shown on the ordinate. The magnitude of hearing loss was most prominent on day 1 and decreased over time to day 7. Of the three test frequencies, the increase in the ABR threshold was the largest at 32 kHz, followed by 16 kHz, and then 8 kHz. The latter findings reflect that auditory sensory cells in the basal turn are very fragile and sensitive to ischemic insult. Administration of BMMCs suppressed the increase in the ABR threshold at all three frequencies, and suppression was most prominent at 32 kHz. The mean ABR threshold increase at 32 kHz on day 1 was 44±7 dB in the BMMC group (n = 6) and 64±6 dB in the saline group (n = 6). Morphological findings

PKH67 staining

The sorted cells were labeled using the fluorescent membrane dye PKH67 (Sigma, Missouri, USA), which excites at a wavelength of 496 nm and emits at 520 nm. According to the manufacturer’s instructions, samples were stained with PKH67 at room temperature for 10 min. Staining was stopped by the addition of four volumes of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. The cells were collected by centrifugation (1500 rpm for 10 min at 41C) and washed twice with DMEM. Tissue preparation for short-term cellular tracking

The gerbils were treated with PKH-labeled BMMCs as described above. Four days after the ischemic insult, they were deeply anesthetized intraperitoneally with a lethal dose of sodium pentobarbital (0.5 g/kg) and were perfused intracardially with saline, followed by 4% paraformaldehyde in PBS. The temporal bones were removed and fixed in the same fixative at 41C for 4 h. In some animals, the fixed temporal bones were decalcified with 0.1 M EDTA for 24 h at 41C and 10-mm-thick cryostat sections of the temporal bone were prepared. The sections were then mounted on 3-aminopropyl triethoxysaline (APS)-coated slide glasses. The sections

Representative photos of the organ of Corti stained with fluorescent dyes are shown in Fig. 2. Nuclei (top) and cellular structures (bottom) of the IHCs sporadically disappeared. Cell loss was minor in the outer hair cells (OHCs). The percentages of hair cell loss are summarized in Fig. 3. In both the BMMC and the saline groups, cell loss was more prominent in IHCs than in OHCs. In IHCs, the percentage of cell loss was 8.8±3.7% in the BMMC group and 19.4±4.1% in the saline group. The difference was statistically significant (P < 0.01). In OHCs, the percentage of cell loss was 4.6±0.7% in the BMMC group and 4.5±1.1% in the saline group; the difference was not significant (P > 0.05). Fate of BMMCs injected into the organ of Corti

We next investigated whether the BMMCs transdifferentiated into cochlear cell types or fused with the injured hair cells after cochlear ischemia. To confirm the fate of BMMCs, we used PKH67 for short-term tracking in vivo. PKH has been used for cellular tracking, and this dye has been shown to be stable on the surface of quiescent cells for periods exceeding 3 weeks, does not compromise cellular viability, and does not impair the capacity of cells to reconstitute hematopoiesis in myeloablated recipients. Using this dye (Fig. 4a), tracking of the BMMCs was



performed at 4 days after ischemia. In a 10-mm-thick cryostat section, the presence of BMMCs was limited to the spiral ganglion area (in the spiral artery of the cochlea; Fig. 4c and d). No PKH-labeled cells were found elsewhere in the organ of Corti (Fig. 4b). These results suggested that, although transplanted BMMCs were retained within the spiral ganglion area of the cochlea,

they were neither transdifferentiated into cochlear cells nor fused with the injured hair cells. Induction of trophic factor after cochlear ischemia

To gain an insight into the mechanisms underlying IHC survival, we investigated the changes in the expression of brain-derived neurotrophic factor (BDNF), VEGF, fibroblast

Fig. 1 Fig. 3

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0 The average increase in the ABR threshold in the bone marrow mononuclear cell (BMMC)-treated group was significantly milder than that in the PBS-treated group. Among the frequencies tested, the higher the frequency, the more severe the magnitude of hearing loss and the differences among the treatment groups. Hearing loss was most prominent on day 1 and decreased in severity over time to day 7 in each frequency. The average ABR threshold shift in the PBS-treated and BMMC-treated groups (n = 6 in each group) was analyzed using the two-tailed Mann–Whitney U-test; *P < 0.05, statistically significant.

Control

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Percentage of IHC and OHC loss at the basal turn. Average cell loss at the basal turn 7 days after ischemia/reperfusion was larger in IHCs than in OHCs. IHC loss was prevented by the administration of BMMCs compared with PBS. All values are presented as the means±SD. Statistical analysis was carried out using the Mann–Whitney U-test; *P < 0.05. BMMC, bone marrow mononuclear cell; IHC, inner hair cell; OHC, outer hair cell.

Fig. 2

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Cochlear hair cells at the basal turn after treatment with PBS or BMMCs. Representative fluorescence images of the organ of Corti stained with rhodamine–phalloidin (a, c) and Hoechst 33342 (b, d). Gerbils were subjected to cochlear ischemia for 7 days. The organs of Corti were obtained from the otic bullae in the PBS-treated group (a, b) and the BMMC-treated group (c, d). Although the nuclei (top) and stereocilia (bottom) of IHCs disappeared sporadically on day 7 in the PBS-treated group (vertical arrowheads), their disappearance was less common in the BMMC-treated group. By contrast, OHCs showed only minor changes after ischemia. Scale bar = 20 mm. BMMC, bone marrow mononuclear cell; IHC, inner hair cell; OHC, outer hair cell.



Fig. 4

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PKH67 labeling. Bone marrow mononuclear cells were labeled with PKH67 (a). Gerbils were treated with PKH67-labeled BMMCs. Four days after ischemia, the temporal bones were dissected, fixed, decalcified, and cut into 10-mm-thick cryostat sections at – 201C. PKH67-positive cells were limited to the spiral ganglion area (in the spiral artery of the cochlea) (c, d). No PKH-labeled cells were found elsewhere in the organ of Corti (b). Scale bars = 50 mm (a), 100 mm (b–d).

growth factor 1, angiopoietin-1, NT-3, and GDNF. Only NT-3 and GDNF protein expression was markedly upregulated by treatment with BMMCs after cochlear ischemia. As shown in Fig. 5a, a single protein band of the expected size for NT-3 (35 kDa) and GDNF (35 kDa) was detected by western blotting with a specific primary antibody. No band was detected when the blots were incubated without the primary antibody (data not shown). Independent experiments were conducted, and the results of densitometric analysis are shown in Fig. 5b. The increase in the NT-3 and GDNF protein level was more prominent in the BMMCtreated group than in the vehicle-treated control. These results showed that NT-3 and GDNF expression was augmented by treatment with BMMCs.

Discussion Idiopathic sudden sensorineural hearing loss is an acute form of hearing loss of unknown etiology involving mainly those aged 40–60 years, and its incidence in Japan is estimated to be 10–20 cases per 100 000 people per year. Although various etiologies have been proposed, acute interruption of the blood supply to the cochlea is considered one of the major causes of sudden deafness [10]. Effective treatment for this condition has, however, been quite limited. Because the Mongolian gerbil lacks the posterior communicating arteries of the circle of Willis, occlusion of the bilateral vertebral artery causes hindbrain ischemia; this model has been used as an animal model of transient cochlear ischemia [15].

We have previously reported the effects of cell-based therapy on the transient cochlear ischemia model by an injection of HSCs through the RW [11]. Most researchers have relied on local application methods through the RW, cochlear wall, posterior semicircular canal, and lateral semicircular canal to provide the inner ears with stem cells. Although direct local application of the stem cells into the cochlea or the vestibule is one of the most reliable delivery methods, it has serious risks of cochlear and/or vestibular functional damage by cochleostomy or labyrinthotomy. We have now shown the usefulness of the systemic delivery of stem cells through the femoral vein to ameliorate ischemic cochlear damage for potential clinical trials. Impairment of cochlear blood flow is considered to play an important role in the etiology of sudden deafness, presbycusis, and noise-induced hearing loss [16]. IHCs are considered to be the main mechanosensory cells that transform mechanical stimuli into neuronal signals [17]. IHC loss started 1 day after ischemia and peaked at 4 days after ischemia, whereas neuronal loss in the spiral ganglion started at 4 days after ischemia and peaked at 7 days after ischemia. These data suggested that ischemiainduced IHC loss resulted in the secondary degeneration of the spiral ganglion neurons [18]. We also showed that progressive IHC loss was closely related to hearing impairment evaluated by ABR [15]. According to these data, if we carried out transplantation therapy with BMMCs on cochlea damaged within a few days after an



Fig. 5

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Western blot analyses of NT-3 and GDNF in gerbil cochleae in the PBS-treated and BMMC-treated groups. The increase in the NT-3 and GDNF protein level was more prominent in the BMMC-treated group than in the PBS-treated control group. Statistical analysis was carried out using one-way analysis of variance, followed by Bonferroni’s multiple comparison test. *P < 0.05, statistically significant; **P < 0.01, highly statistically significant than the PBS-treated control. All values are presented as means±SD. BMMC, bone marrow mononuclear cell; GDNF, glial cell line-derived neurotrophic factor; NT-3, neurotrophin-3.

ischemic insult, most of the IHCs may be rescued from degeneration. Cell transplantation using bone marrow mononuclear cells (BMMCs) has been shown to accelerate angiogenesis/neovascularization in several ischemic diseases, such as limb ischemia [4] and myocardial infarction [3]. BMMCs contain EPCs that have been shown to contribute toward revascularization of ischemic tissues and repair of injured endothelium [2]. Furthermore, BMMCs contain several types of bone marrow cells, including HSCs and mesenchymal stem cells (MSCs). These stem cells have been reported to secrete multiple growth factors, such as VEGF, glia-derived neurotrophic factor (GDNF), BDNF, nerve growth factor, and hepatocyte growth factor [19,20]. In addition, BMMCs have been considered to be responsible for angiogenic, antiapoptotic, and mitogenic effects once transplanted into the myocardium and brain [1,21]. In the present study, we showed that systemic administration of BMMCs after ischemia ameliorated progressive IHC damage and prevented a shift in the ABR threshold. Using the PKH67 dye, the presence of BMMCs was limited in the spiral artery of the cochlea. No PKHlabeled cells were found elsewhere in the organ of Corti. These results suggested that, although transplanted BMMCs were retained within the spiral ganglion area of

the cochlea, they were neither transdifferentiated into cochlear cells nor fused with the injured hair cells and supporting cells in the organ of Corti to restore their functions. The possibilities of functional recovery by treatment with BMMCs are that BMMCs can promote hair cell repair, in part, by secreting trophic factors. It has been reported that trophic factors can confer resistance to disease or promote the survival, migration, and differentiation of endogenous precursors [1,7,19–21]. Bone marrow cell transplantation may be linked to the upregulation of trophic factors [1]. Several types of trophic factors play a crucial role in the survival of sensory hair cells and auditory neurons [22]. Previously, we have reported that an intrascalar injection of HSCs prevents hair cell death after transient cochlear ischemia through paracrine effects. HSCs showed the potential to upregulate the GDNF protein level in the organ of Corti after ischemia [11]. We also showed that adenovirus-mediated overexpression of GDNF significantly prevented progressive IHC degeneration after cochlear ischemia [23], and insulin-like growth factor 1 treatment by hydrogels rescues cochlear hair cells from ischemic injury [24]. In the present study, we evaluated the ischemia-induced alterations of trophic factors (i.e. VEGF, FGF-2, BDNF, GDNF, NT-3, and Ang1) in the cochlea. Consequently, we found that only NT-3 and GDNF expression was



upregulated after cochlear ischemia, and this ischemiainduced trophic factor expression was augmented by treatment with BMMCs. We suggest that BMMCs have therapeutic potential, possibly through paracrine effects. Therefore, we propose using BMMCs as a potential new therapeutic strategy for hearing loss, although further cellular and molecular biological investigations are required to clarify its mechanism. Several in-vivo studies have shown that the degeneration in mature cochlea could be reduced if trophic factors are provided to the spiral ganglion neurons [25]. Ischemiainduced IHC loss resulted in the secondary degeneration of the spiral ganglion neurons [18]. We are planning to elucidate whether trophic factors expression by BMMCs could attenuate the degeneration of SG neurons, although further histochemical investigation is required to confirm this assumption.

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Conclusion Our study clearly showed that an intravenous injection of BMMCs prevented a shift in the ABR threshold and attenuated the progressive IHC damage after cochlear ischemia. We also observed the migration of systemically infused BMMCs into seriously injured cochlea. Injected BMMCs had the potential to upregulate the protein level of trophic factors (NT-3, GDNF) in the organ of Corti after cochlear ischemia. The long-term effects of BMMC transplantation have not been elucidated; however, these data suggest that systemic delivery of BMMCs may be another good option for cell-based therapy of hearing loss.

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Acknowledgements

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This study was supported by Grants-in-Aid for Scientific Research (23592486).

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Conflicts of interest

There are no conflicts of interest.

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Autologous bone marrow mononuclear cells intrathecal transplantation in chronic stroke.

Designer aminoglycosides prevent cochlear hair cell loss and hearing loss.

Bone marrow-derived mononuclear cells do not exert acute neuroprotection after stroke in spontaneously hypertensive rats.

Proliferative and interferon responses by peripheral blood mononuclear cells after bone marrow transplantation in humans.

Effects of bone marrow mesenchymal stem cells (BM-MSCs) on rat pial microvascular remodeling after transient middle cerebral artery occlusion.

EMMPRIN regulates tumor growth and metastasis by recruiting bone marrow-derived cells through paracrine signaling of SDF-1 and VEGF.

Effects of mTOR on Neurological Deficits after Transient Global Ischemia.

Characterization of bone marrow mononuclear cells on biomaterials for bone tissue engineering in vitro.

Corrigendum to "Characterization of Bone Marrow Mononuclear Cells on Biomaterials for Bone Tissue Engineering In Vitro".

Intramuscular injection of bone marrow mononuclear cells contributes to bone repair following midpalatal expansion in rats.

Accelerated functional recovery after skeletal muscle ischemia-reperfusion injury using freshly isolated bone marrow cells.

prostacyclin pathway in pulmonary arterial hypertension.