Deletion of C1ql1 Causes Hearing Loss and Abnormal Auditory Nerve Fibers in the Mouse Cochlea.

Complement C1q Like 1 (C1QL1), a secreted component of C1Q-related protein, is known to play an important role in synaptic maturation, regulation, and maintenance in the central nervous system. C1ql1 is expressed in adult cochlear inner and outer hair cells (IHCs and OHCs) with preferential expression in OHCs. We generated C1ql1 null mice to examine the role of C1QL1 in the auditory periphery. C1ql1-null mice exhibited progressive hearing loss with elevated thresholds of auditory brainstem response and distortion product otoacoustic emission. Confocal microscopy showed that the number of nerve fibers innervating both IHCs and OHCs was significantly reduced. However, spiral ganglion neurons appeared to be normal under electron microscopy. IHC development and survival were not affected by deletion of C1ql1. Voltage-clamp recording and immunocytochmistry combined with confocal microscopy showed C1ql1-null IHCs showed no significant reduction of pre-synaptic proteins and synaptic vesicle release. This is in contrast to significant OHC loss in the KO mice. Our study suggests that C1ql1 is essential for development of hair cell innervation and OHC survival. But maturation of presynaptic machinery in IHCs does not depend on C1QL1.

Loss of cochlear ribbon synapses in the early stage of aging causes initial hearing impairment.

Hearing loss can occur with aging. However, there remains debate about which cochlear component is the most susceptible to aging insult and the consequent pathological events responsible for age-related hearing loss. In this study, we used C57BL/6J mice to mimic the process of aging, and the auditory brainstem response (ABR) thresholds of aging mice were examined at different stages of aging (1, 2, 4, and 6 months [M]). The lifespan of 4 M was considered to be the early stage of aging. Immunostaining combined with laser confocal microscopy was employed to identify RIBEYE/CtBP2, a marker of cochlear ribbon synapses, and a quantitative analysis of the synaptic ribbon was carried out. The function of the ribbon synapse was estimated by amplitude alterations of ABR wave I. Furthermore, endocytosis of the inner hair cells was also detected using the fluorescence labeling dye FM1-43. We found that the loss of ribbon synapses in the early stage of aging occurred prior to hair cell or auditory nerve loss and was the initial pathological change. Additionally, the loss of ribbon synapses, including the quantity and function of synapses, was found to correspond to the elevations of the hearing threshold across frequencies. Moreover, a significant reduction in the endocytosis function of the inner hair cells was identified in the early stage of aging. Therefore, our study indicated that the reduction of cochlear ribbon synapses occurred at an early stage of aging and could be responsible for the consequent hearing loss.

Loss of Cochlear Ribbon Synapse Is a Critical Contributor to Chronic Salicylate Sodium Treatment-Induced Tinnitus without Change Hearing Threshold.

Tinnitus is a common auditory disease worldwide; it is estimated that more than 10% of all individuals experience this hearing disorder during their lifetime. Tinnitus is sometimes accompanied by hearing loss. However, hearing loss is not acquired in some other tinnitus generations. In this study, we injected adult rats with salicylate sodium (SS) (200 mg/kg/day for 10 days) and found no significant hearing threshold changes at 2, 4, 8, 12, 14, 16, 20, or 24 kHz (all p > 0.05). Tinnitus was confirmed in the treated rats via Behaviour Testing of Acoustic Startle Response (ASR) and Gap Prepulse Inhibition Test of Acoustic Startle Reflex (GPIAS). A immunostaining study showed that there is significant loss of anti-CtBP2 puncta (a marker of cochlear inner hair cell (HC) ribbon synapses) in treated animals in apical, middle, and basal turns (all p < 0.05). The ABR wave I amplitudes were significantly reduced at 4, 8, 12, 14, 16, and 20 kHz (all p < 0.05). No significant losses of outer HCs, inner HCs, or HC cilia were observed (all p > 0.05). Thus, our study suggests that loss of cochlear inner HC ribbon synapse after SS exposure is a contributor to the development of tinnitus without changing hearing threshold.

Repeated Moderate Sound Exposure Causes Accumulated Trauma to Cochlear Ribbon Synapses in Mice.

Repeated induction of a temporary threshold shift (TTS) may result in a permanent threshold shift (PTS) and is thought to be associated with early onset of age-related hearing loss (ARHL). The possibility that a PTS might be induced by administration of repeated TTS-inducing noise exposures (NEs) over a short period during early adulthood has not been formally investigated. We aimed to investigate possible cumulative acoustic overstimulation effects that permanently shift the auditory threshold. Young adult C57BL/6J mice were exposed twice to moderate white noise in an experimental design that minimized the effects of aging. The first exposure resulted in a reversible noise-induced hearing loss (NIHL) measured as recoverable alterations in auditory brainstem response (ABR) thresholds, waveform amplitudes, and numbers of ribbon synapses. The second NE with the same parameters caused persistent threshold shifts, wave I amplitude reductions, wave IV/I ratio enhancements, and synaptic losses, even though recovery time sufficient for a TTS had been provided. The pattern of PTS resembled NIHL since the observed impairments tonotopically followed the power spectrum of the noise insult, rather than ARHL, which distributes at higher frequencies. No significant changes were observed in the control group as the mice aged. To conclude, our results demonstrate a cumulative effect of repetitive TTS-inducing NE on hearing function and synaptic plasticity that does not cause premature ARHL, thereby providing insight into the pathophysiological mechanisms underlying NIHL and ARHL.

Age-related insult of cochlear ribbon synapses: An early-onset contributor to D-galactose-induced aging in mice.

Presbycusis results from age-related degeneration of the auditory system. D-galactose (D-gal)-induced aging is an ideal and commonly used animal model in aging research. Previous studies demonstrate that administration of D-gal can activate mitochondria-dependent apoptosis in the cochlear stria vascularis. However, D-gal-induced changes to cochlear inner (IHCs) and outer (OHCs) hair cells, spiral ganglion cells (SGCs), and ribbon synapses connecting IHCs and SGCs have not been systematically reported. The current study investigated changes in the numbers of hair cells, SGCs, and ribbon synapses in the mouse model of aging. We found that in comparison to control mice, the numbers of ribbon synapses and their nerve fibers were significantly decreased in D-gal-treated mice, whereas the numbers of OHCs, IHCs, and SGCs were almost unchanged. Moreover, hair cell stereocilia were also not obviously influenced by D-gal administration. Although D-gal-induced aging did not significantly shift the auditory brainstem response (ABR) thresholds in the 8, 16, and 32 kHz frequency bands, the amplitude and latency of the ABR wave I, reflecting ribbon synapse functions, were abnormal in D-gal-treated mice compared to control mice. We also found that 8-hydroxy-2-deoxyguanosine, a marker of oxidative DNA damage, was significantly increased in mitochondria of cochleae from mice exposed to D-gal-induced aging in comparison to control mice. Moreover, D-gal administration increased the levels of H2O2 and mitochondrial 3860-bp common deletion, and decreased superoxide dismutase activity and ATP production in the cochlea. Furthermore, compared with control mice, the protein levels of NADPH oxidase 2 and uncoupling protein 2 were significantly increased in the cochlea of D-gal-treated mice. Taken together, these findings support that the cochlear ribbon synapse is the primary insult site in the early stage of presbycusis, and mitochondrial oxidative damage and subsequent dysfunctions might be responsible for this insult.

NADPH Oxidase 2-Mediated Insult in the Auditory Cortex of Zucker Diabetic Fatty Rats.

Clinical data has confirmed that auditory impairment may be a secondary symptom of type 2 diabetes mellitus (T2DM). However, mechanisms underlying pathologic changes that occur in the auditory system, especially in the central auditory system (CAS), remain poorly understood. In this study, Zucker diabetic fatty (ZDF) rats were used as a T2DM rat model to observe ultrastructural alterations in the auditory cortex and investigate possible mechanisms underlying CAS damage in T2DM. The auditory brainstem response (ABR) of ZDF rats was found to be markedly elevated in low (8 kHz) and high (32 kHz) frequencies. Protein expression of NADPH oxidase 2 (NOX2) and its matching subunits P22phox, P47phox, and P67phox was increased in the auditory cortex of ZDF rats. Expression of 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of DNA oxidative damage, was also increased in the neuronal mitochondria of the auditory cortex of ZDF rats. Additionally, decreases in the mitochondrial total antioxidant capabilities (T-AOC), adenosine triphosphate (ATP) production, and mitochondrial membrane potential (MMP) were detected in the auditory cortex of ZDF rats, suggesting mitochondrial dysfunction. Transmission electron microscopy results indicated that ultrastructural damage had occurred to neurovascular units and mitochondria in the auditory cortex of ZDF rats. Furthermore, cytochrome c (Cyt c) translocation from mitochondria to cytoplasm and caspase 3-dependent apoptosis were also detected in the auditory cortex of ZDF rats. Consequently, the study demonstrated that T2DM may cause morphological damage to the CAS and that NOX2-associated mitochondrial oxidative damage and apoptosis may be partly responsible for this insult.

Different patterns of endocytosis in cochlear inner and outer hair cells of mice.

Precise and efficient endocytosis is critical for sustained neurotransmission during continuous neuronal activity. Endocytosis is a prerequisite for maintaining the auditory function. However, the differences between the patterns of endocytosis in cochlear inner hair cells (IHCs) and outer hair cells (OHCs) remain unclear. Both IHCs and OHCs were obtained from adult C57 mice. Patterns of endocytosis in cells were estimated by analyzing the uptake of FM1-43, a fluorescent. The observations were made using live confocal imaging, fluorescence intensities were calculated statistically. Results revealed the details about following phenomenon, i) sites of entry: the FM1-43 dye was found to enter IHC at the apical area initially, the additional sites of entry were then found at basolateral membrane of the cells, The entry of the dye into OHCs initially appeared to be occurring around whole apical membranes area, which then diffused towards the other membrane surface of the cells, ii) capacity of endocytosis: fluorescence intensity in IHCs showed significantly higher than that of OHCs (P<0.01). We have found different patterns of endocytosis between IHCs and OHCs, this indicated functional distinctions between them. Moreover, FM1-43 dye can be potentially used as an indicator of the functional loss or repair of cochlear hair cells.

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea.

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear "synaptopathy", which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

Mitochondrial DNA 3,860-bp Deletion Increases with Aging in the Auditory Nervous System of C57BL/6J Mice.

BACKGROUND/AIMS: Mitochondrial DNA (mtDNA) is sensitive to oxidative damage during aging, which can result in mtDNA mutations. A previous study reported that a 3,860-bp mtDNA deletion, similar to a 4,977-bp mtDNA deletion in humans, is also common occurrence in murine tissues, and increases in the brain and liver with aging. However, no previous study evaluated both topics in the murine auditory nervous system. METHODS: We compared mtDNA oxidative damage, mitochondrial ultrastructural changes, and the frequency of the 3,860-bp deletion in the peripheral (spiral ganglion, SG) and central (auditory cortex, AC) auditory nervous system of C57BL/6J mice aged 2, 12, and 18 months. RESULTS: We found that the threshold of auditory brainstem response increased with age along with the signal of 8-hydroxy-2'-deoxyguanosine - a marker of DNA oxidative damage - in the mitochondria of SG and AC neurons. The mitochondrial ultrastructural damage also increased with aging in the SG and AC neurons. Moreover, the relative amount of mtDNA 3,860-bp deletion in 12- and 18-month-old mice was significantly higher in the SG and AC as compared to 2-month-old mice. CONCLUSION: These results suggest that the mtDNA 3,860-bp deletion is common in the auditory nervous system of mice and increases with age and may contribute to age-related hearing loss.

Dynamic Changes of Functional Neuronal Activities Between the Auditory Pathway and Limbic Systems Contribute to Noise-Induced Tinnitus with a Normal Audiogram.

Tinnitus is thought to be triggered by aberrant neural activity in the central auditory pathway and is often accompanied by comorbidities of emotional distress and anxiety, which imply maladaptive functional connectivity to limbic structures, such as the amygdala and hippocampus. Tinnitus patients with normal audiograms can also have accompanying anxiety and depression, clinically. To test the role of functional connectivity between the central auditory pathway and limbic structures in patients with tinnitus with normal audiograms, we developed a murine noise-induced tinnitus model with a temporary threshold shift (TTS). Tinnitus mice exhibited reduced auditory brainstem response wave I amplitude, and an enhanced wave IV amplitude and wave IV/I amplitude ratio, as compared with control and non-tinnitus mice. Resting-state functional magnetic resonance imaging (fMRI) was used to identify abnormal connectivity of the amygdala and hippocampus and to determine the relationship with tinnitus characteristics. We found increased fMRI responses with amplitude of low-frequency fluctuation (ALFF) in the auditory cortex and decreased ALFF in the amygdala and hippocampus at day 1, but decreased ALFF in the auditory cortex and increased ALFF in the amygdala at day 28 post-noise exposure in tinnitus mice. Decreased functional connectivity between auditory brain regions and limbic structures was demonstrated at day 28 in tinnitus mice. Therefore, aberrant neural activities in tinnitus mice with TTS involved not only the central auditory pathway, but also limbic structures, and there was maladaptive functional connectivity between the central auditory pathway and limbic structures, such as the amygdala and hippocampus.

Long-Term Conductive Auditory Deprivation During Early Development Causes Irreversible Hearing Impairment and Cochlear Synaptic Disruption.

Conductive hearing loss is a prevalent condition globally. It remains unclear whether conductive hearing loss that occurs during early development disrupts auditory peripheral systems. In this study, a mouse model of conductive auditory deprivation (CAD) was achieved using external auditory canal closure on postnatal day 12, which marks the onset of external ear canal opening. Short-term (2 weeks) and long-term (6 weeks) deprivations involving external ear canal closure were conducted. Mice were examined immediately, 4 weeks, and 8 weeks after deprivation. Short-term deprivation induced reversible auditory brainstem response (ABR) threshold and latencies of ABR wave I, whereas long-term deprivation caused irreversible ABR thresholds and latencies of ABR wave I. Complete recovery of ribbon synapses and latencies of ABR wave I was observed in the short-term group. In contrast, we observed irreversible ABR thresholds, latencies of ABR wave I, and quantity of ribbon synapses in the long-term deprivation group. Positive 8-hydroxy-2'-deoxyguanosine signals were noted in cochlear hair cells in the long-term group, suggesting that long-term auditory deprivation could disrupt auditory maturation via mitochondrial damage in cochlear hair cells. Conversely, no significant changes in cellular morphology were observed in cochlear hair cells and spiral ganglion cells in either short- or long-term groups. Collectively, our findings suggest that long-term conductive hearing deprivation during early stages of auditory development can cause significant and irreversible disruption that persists into adulthood.

NADPH oxidase inhibitor apocynin decreases mitochondrial dysfunction and apoptosis in the ventral cochlear nucleus of D-galactose-induced aging model in rats.

Presbycusis has become a common sensory deficit in humans. Oxidative damage to mitochondrial DNA and mitochondrial dysfunction is strongly associated with the aging of the auditory system. A previous study established a mimetic rat model of aging using D-galactose (D-gal) and first reported that NADPH oxidase-dependent mitochondrial oxidative damage and apoptosis in the ventral cochlear nucleus (VCN) might contribute to D-gal-induced central presbycusis. In this study, we investigated the effects of apocynin, an NADPH oxidase inhibitor, on mitochondrial dysfunction and mitochondria-dependent apoptosis in the VCN of D-gal-induced aging model in rats. Our data showed that apocynin decreased NADPH oxidase activity, H2O2 levels, mitochondrial DNA common deletion, and 8-hydroxy-2-deoxyguanosine (8-OHdG) expression and increased total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activity in the VCN of D-gal-induced aging model in rats. Moreover, apocynin also decreased the protein levels of phospho-p47phox (p-p47phox), tumor necrosis factor alpha (TNFα), and uncoupling protein 2 (UCP2) in the VCN of D-gal-induced aging model in rats. Meanwhile, apocynin alleviated mitochondrial ultrastructure damage and enhanced ATP production and mitochondrial membrane potential (MMP) levels in the VCN of D-gal-induced aging model in rats. Furthermore, apocynin inhibited cytochrome c (Cyt c) translocation from mitochondria to the cytoplasm and suppressed caspase 3-dependent apoptosis in the VCN of D-gal-induced aging model in rats. Consequently, our findings suggest that neuronal survival promoted by an NADPH oxidase inhibitor is a potentially effective method to enhance the resistance of neurons to central presbycusis.

Insulin-like growth factor binding protein 4 inhibits proliferation of bone marrow mesenchymal stem cells and enhances growth of neurospheres derived from the stem cells.

Insulin-like growth factor binding protein 4 (IGFBP-4) was reported to trigger cellular senescence and reduce cell growth of bone marrow mesenchymal stem cells (BMSCs), but its contribution to neurogenic differentiation of BMSCs remains unknown. In the present study, BMSCs were isolated from the femur and tibia of young rats to investigate effects of IGFBP-4 on BMSC proliferation and growth of neurospheres derived from BMSCs. Bone marrow mesenchymal stem cell proliferation was assessed using CCK-8 after treatment with IGFBP-4 or blockers of IGF-IR and ß-catenin. Phosphorylation levels of Akt, Erk, and p38 in BMSCs were analysed by Western blotting. Bone marrow mesenchymal stem cells were induced into neural lineages in NeuroCult medium; the number and the size of BMSC-derived neurospheres were counted after treatment with IGFBP-4 or the blockers. It was shown that addition of IGFBP-4 inhibited BMSC proliferation and immunodepletion of IGFBP-4 increased the proliferation. The blockade of IGF-IR with AG1024 increased BMSC proliferation and reversed IGFBP-4-induced proliferation inhibition; however, blocking of ß-catenin with FH535 did not. p-Erk was significantly decreased in IGFBP-4-treated BMSCs. IGFBP-4 promoted the growth of neurospheres derived from BMSCs, as manifested by the increases in the number and the size of the derived neurospheres. Both AG1024 and FH535 inhibited the formation of NeuroCult-induced neurospheres, but FH535 significantly inhibited the growth of neurospheres in NeuroCult medium with EGF, bFGF, and IGFBP-4. The data suggested that IGFBP-4 inhibits BMSC proliferation through IGF-IR pathway and promotes growth of BMSC-derived neurospheres via stabilizing ß-catenin.

Isolation and characterization of endothelial colony-forming cells from mononuclear cells of rat bone marrow.

Transplantation of bone marrow-derived endothelial progenitor cells (BM-EPCs) has been used as a therapeutic strategy for vascular repair. However, it remains controversial whether BM-EPCs exhibit clonal endothelial colony-forming cell (ECFC) capacity, a characteristic of true EPCs. The aim of this study was to isolate and explore the cellular properties of BM-ECFCs. We isolated BM-ECFCs from rat bone marrow with high purity via an optimized method. This approach involved the removal of selective colonies based on the conventional differential adhesive culture method used to isolate ECFCs from peripheral and umbilical cord blood. Our results indicate that primary colony BM-ECFCs display a panel of surface antigen markers consistent with endothelial cells. These BM-ECFCs coexpress CD34, CD133, and VEGFR2 at high levels, and these levels decrease with passaging. These cells have high potential for proliferation, migration, and formation of capillary-like structures on Matrigel, and these abilities are retained during ex vivo expansion. Furthermore, BM-ECFCs cultured with 10% or 20% fetal bovine serum demonstrated two different patterns of spontaneous capillary-like structure formation. These results provide a foundation for isolation of ECFCs from human bone marrow for autologous cell transplantation and tissue engineering applications in the future.

Hierarchically aligned fibrin nanofiber hydrogel accelerated axonal regrowth and locomotor function recovery in rat spinal cord injury.

BACKGROUND: Designing novel biomaterials that incorporate or mimic the functions of extracellular matrix to deliver precise regulatory signals for tissue regeneration is the focus of current intensive research efforts in tissue engineering and regenerative medicine. METHODS AND RESULTS: To mimic the natural environment of the spinal cord tissue, a three-dimensional hierarchically aligned fibrin hydrogel (AFG) with oriented topography and soft stiffness has been fabricated by electrospinning and a concurrent molecular self-assembling process. In this study, the AFG was implanted into a rat dorsal hemisected spinal cord injury model to bridge the lesion site. Host cells invaded promptly along the aligned fibrin hydrogels to form aligned tissue cables in the first week, and then were followed by axonal regrowth. At 4 weeks after the surgery, neurofilament (NF)-positive staining fibers were detected near the rostral end as well as the middle site of defect, which aligned along the tissue cables. Abundant NF- and GAP-43-positive staining indicated new axon regrowth in the oriented tissue cables, which penetrated throughout the lesion site in 8 weeks. Additionally, the abundant blood vessels marked with RECA-1 had reconstructed within the lesion site at 4 weeks after surgery. Basso-Beattie-Bresnahan scoring showed that the locomotor performance of the AFG group recovered much faster than that of blank control group or the random fibrin hydrogel (RFG) group from 2 weeks after surgery. Furthermore, diffusion tensor imaging tractography of MRI confirmed the optimal axon fiber reconstruction compared with the RFG and control groups. CONCLUSION: Taken together, our results suggested that the AFG scaffold provided an inductive matrix for accelerating directional host cell invasion, vascular system reconstruction, and axonal regrowth, which could promote and support extensive aligned axonal regrowth and locomotor function recovery.

The histone deacetylase inhibitor sodium butyrate protects against noise-induced hearing loss in Guinea pigs.

Noise-induced hearing loss (NIHL) severely impacts the quality of life of affected individuals. Oxidative stress resulting from noise exposure is a significant cause of NIHL. Although histone deacetylase (HDAC) inhibitors were shown to protect against NIHL, the underlying mechanism remains unclear, and it is not known how they act on noise-induced oxidative stress. In the current study, we investigated the expression levels of acetyl-histone H3 (Lys9) (H3-AcK9), histone deacetylase 1 (HDAC1), and 3-nitrotyrosine (3-NT), an oxidative stress marker, in a guinea pig model of NIHL using immunohistology and Western blotting. We then assessed the effects of systemic administration of the HDAC inhibitor, sodium butyrate (SB), on noise-induced permanent threshold shifts (PTS), hair cell (HC) loss, and changes in the above mentioned markers. The results showed that SB attenuated noise-induced PTS and outer hair cell loss. SB treatment promoted H3-AcK9 expression and repressed HDAC1 expression in the nuclei of HCs and Hensen's cells after noise exposure. Furthermore, SB attenuated the noise-induced increase of 3-NT expression in HCs and Hensen's cells. These findings suggest that SB protects against NIHL by reversing the noise-induced histone acetylation imbalance and inhibiting oxidative stress in cochlear HCs and Hensen's cells. SB treatment may represent a potential strategy to prevent and treat NIHL.

Drug-nanoencapsulated PLGA microspheres prepared by emulsion electrospray with controlled release behavior.

The development of modern therapeutics has raised the requirement for controlled drug delivery system which is able to efficiently encapsulate bioactive agents and achieve their release at a desired rate satisfying the need of the practical system. In this study, two kind of aqueous model drugs with different molecule weight, Congo red and albumin from bovine serum (BSA) were nano-encapsulated in poly (dl-lactic-co-glycolic acid) (PLGA) microspheres by emulsion electrospray. In the preparation process, the aqueous phase of drugs was added into the PLGA chloroform solution to form the emulsion solution. The emulsion was then electrosprayed to fabricate drug-nanoencapsulated PLGA microspheres. The morphology of the PLGA microspheres was affected by the volume ratio of aqueous drug phase and organic PLGA phase (Vw/Vo ) and the molecule weight of model drugs. Confocal laser scanning microcopy showed the nanodroplets of drug phase were scattered in the PLGA microspheres homogenously with different distribution patterns related to Vw/Vo . With the increase of the volume ratio of aqueous drug phase, the number of nanodroplets increased forming continuous phase gradually that could accelerate drug release rate. Moreover, BSA showed a slower release rate from PLGA microspheres comparing to Congo red, which indicated the drug release rate could be affected by not only Vw/Vo but also the molecule weight of model drug. In brief, the PLGA microspheres prepared using emulsion electrospray provided an efficient and simple system to achieve controlled drug release at a desired rate satisfying the need of the practices.

Angiogenic microspheres promote neural regeneration and motor function recovery after spinal cord injury in rats.

This study examined sustained co-delivery of vascular endothelial growth factor (VEGF), angiopoietin-1 and basic fibroblast growth factor (bFGF) encapsulated in angiogenic microspheres. These spheres were delivered to sites of spinal cord contusion injury in rats, and their ability to induce vessel formation, neural regeneration and improve hindlimb motor function was assessed. At 2-8 weeks after spinal cord injury, ELISA-determined levels of VEGF, angiopoietin-1, and bFGF were significantly higher in spinal cord tissues in rats that received angiogenic microspheres than in those that received empty microspheres. Sites of injury in animals that received angiogenic microspheres also contained greater numbers of isolectin B4-binding vessels and cells positive for nestin or ß III-tubulin (P < 0.01), significantly more NF-positive and serotonergic fibers, and more MBP-positive mature oligodendrocytes. Animals receiving angiogenic microspheres also suffered significantly less loss of white matter volume. At 10 weeks after injury, open field tests showed that animals that received angiogenic microspheres scored significantly higher on the Basso-Beattie-Bresnahan scale than control animals (P < 0.01). Our results suggest that biodegradable, biocompatible PLGA microspheres can release angiogenic factors in a sustained fashion into sites of spinal cord injury and markedly stimulate angiogenesis and neurogenesis, accelerating recovery of neurologic function.

Co-effects of matrix low elasticity and aligned topography on stem cell neurogenic differentiation and rapid neurite outgrowth.

The development of novel biomaterials that deliver precise regulatory signals to direct stem cell fate for nerve regeneration is the focus of current intensive research efforts. In this study, a hierarchically aligned fibrillar fibrin hydrogel (AFG) that was fabricated through electrospinning and the concurrent molecular self-assembly process mimics both the soft and oriented features of nerve tissue, thus providing hybrid biophysical cues to instruct cell behavior in vitro and in vivo. The electrospun hydrogels were examined by scanning electron microscopy (SEM), polarized light microscopy, small angle X-ray scattering assay and atomic force microscopy (AFM), showing a hierarchically linear-ordered structure from the nanoscale to the macroscale with a soft elastic character (elasticity ∼1 kPa). We found that this low elasticity and aligned topography of AFG exhibit co-effects on promoting the neurogenic differentiation of human umbilical cord mesenchymal stem cells (hUMSCs) in comparison to random fibrin hydrogel (RFG) and tissue culture plate (TCP) control after two week cell culture in growth medium lacking supplementation with soluble neurogenic induction factors. In addition, AFG also induces dorsal root ganglion (DRG) neurons to rapidly project numerous long neurite outgrowths longitudinally along the AFG fibers for a total neurite extension distance of 1.96 mm in three days in the absence of neurotrophic factor supplementation. Moreover, the AFG implanted in a rat T9 dorsal hemisection spinal cord injury model was found to promote endogenous neural cell fast migration and axonal invasion along AFG fibers, resulting in aligned tissue cables in vivo. Our results suggest that matrix stiffness and aligned topography may instruct stem cell neurogenic differentiation and rapid neurite outgrowth, providing great promise for biomaterial design for applications in nerve regeneration.

Spinal cord injury repair by implantation of structured hyaluronic acid scaffold with PLGA microspheres in the rat.

In order to create an optimal microenvironment for neural regeneration in the lesion area after spinal cord injury (SCI), we fabricated a novel scaffold composed of a hyaluronic acid (HA) hydrogel with a longitudinal multi-tubular conformation. The scaffold was modified by binding with an anti-Nogo receptor antibody (antiNgR) and mixed further with poly(lactic-co-glycolic acid) (PLGA) microspheres containing brain-derived neurotrophic factor and vascular endothelial growth factor (HA+PLGA). In the rat, after implantation of this composite into an injured area created by a dorsal hemisection at T9-10 of the spinal cord, favorable effects were seen with regard to the promotion of spinal repair, including excellent integration of the implants with host tissue, inhibition of inflammation, and gliosis. In particular, large numbers of new blood vessels and regenerated nerve fibers were found within and around the implants. Simultaneously, the implanted rats exhibited improved locomotor recovery. Thus, this novel composite material might provide a suitable microenvironment for neural regeneration following SCI.