Improvement of Sciatic Nerve Regeneration Using Laminin-Binding Human NGF-β

Background

Sciatic nerve injuries often cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life. Nerve growth factor (NGF) plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical applications. We reported here by fusing with the N-terminal domain of agrin (NtA), NGF-β could target to nerve cells and improve nerve regeneration.

Methods

Laminin-binding assay and sustained release assay of NGF-β fused with NtA (LBD-NGF) from laminin in vitro were carried out. The bioactivity of LBD-NGF on laminin in vitro was also measured. Using the rat sciatic nerve crush injury model, the nerve repair and functional restoration by utilizing LBD-NGF were tested.

Findings

LBD-NGF could specifically bind to laminin and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that LBD-NGF could be retained and concentrated at the nerve injury sites to promote nerve repair and enhance functional restoration following nerve damages.

Conclusion

Fused with NtA, NGF-β could bind to laminin specifically. Since laminin is the major component of nerve extracellular matrix, laminin binding NGF could target to nerve cells and improve the repair of peripheral nerve injuries.     

Allografts of the acellular sciatic nerve and brain-derived neurotrophic factor repair spinal cord injury in adult rats

Objective

We aimed to investigate whether an innovative growth factor-laden scaffold composed of acellular sciatic nerve (ASN) and brain-derived neurotrophic factor (BDNF) could promote axonal regeneration and functional recovery after spinal cord injury (SCI).

Methods

Following complete transection at the thoracic level (T9), we immediately transplanted the grafts between the stumps of the severed spinal cords. We evaluated the functional recovery of the hindlimbs of the operated rats using the BBB locomotor rating scale system every week. Eight weeks after surgery, axonal regeneration was examined using the fluorogold (FG) retrograde tracing method. Electrophysiological analysis was carried out to evaluate the improvement in the neuronal circuits. Immunohistochemistry was employed to identify local injuries and recovery.

Results

The results of the Basso-Beattie-Bresnahan (BBB) scale indicated that there was no significant difference between the individual groups. The FG retrograde tracing and electrophysiological analyses indicated that the transplantation of ASN-BDNF provided a permissive environment to support neuron regeneration.

Conclusion

The ASN-BDNF transplantation provided a promising therapeutic approach to promote axonal regeneration and recovery after SCI, and can be used as part of a combinatory treatment strategy for SCI management.     

Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep

Background

Surgical reapposition of peripheral nerve results in some axonal regeneration and functional recovery, but the clinical outcome in long distance nerve defects is disappointing and research continues to utilize further interventional approaches to optimize functional recovery. We describe the use of nerve constructs consisting of decellularized vein grafts filled with spider silk fibers as a guiding material to bridge a 6.0 cm tibial nerve defect in adult sheep.

Methodology/Principal Findings

The nerve constructs were compared to autologous nerve grafts. Regeneration was evaluated for clinical, electrophysiological and histological outcome. Electrophysiological recordings were obtained at 6 months and 10 months post surgery in each group. Ten months later, the nerves were removed and prepared for immunostaining, electrophysiological and electron microscopy. Immunostaining for sodium channel (NaV 1.6) was used to define nodes of Ranvier on regenerated axons in combination with anti-S100 and neurofilament. Anti-S100 was used to identify Schwann cells. Axons regenerated through the constructs and were myelinated indicating migration of Schwann cells into the constructs. Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons. There was no significant difference in electrophysiological results between control autologous experimental and construct implantation indicating that our construct are an effective alternative to autologous nerve transplantation.

Conclusions/Significance

This study demonstrates that spider silk enhances Schwann cell migration, axonal regrowth and remyelination including electrophysiological recovery in a long-distance peripheral nerve gap model resulting in functional recovery. This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.     

Omentum-wrapped scaffold with longitudinally oriented micro-channels promotes axonal regeneration and motor functional recovery in rats

Background

Tissue-engineered nerve scaffolds hold great potential in bridging large peripheral nerve defects. However, insufficient vascularization of nerve scaffolds limited neural tissues survival and regeneration, which hampered the successful implantation and clinical application of nerve scaffolds. The omentum possesses a high vascularization capacity and enhances regeneration and maturation of tissues and constructs to which it is applied. However, combined application of nerve scaffolds and omentum on axonal regeneration and functional recovery in the treatment of large peripheral nerve defects has rarely been investigated thus far.

Methods

In the present study, an omentum-wrapped collagen-chitosan scaffold was used to bridge a 15-mm-long sciatic nerve defect in rats. Rats that received nerve autografts or scaffolds alone were served as positive control or negative control, respectively. The axonal regeneration and functional recovery were examined by a combination of walking track analysis, electrophysiological assessment, Fluoro-Gold (FG) retrograde tracing, as well as morphometric analyses to both regenerated nerves and target muscles.

Findings

The results demonstrated that axonal regeneration and functional recovery were in the similar range between the omentum-wrapping group and the autograft group, which were significantly better than those in the scaffold alone group. Further investigation showed that the protein levels of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were significantly higher in the omentum-wrapping group than those in the scaffold alone group in the early weeks after surgery.

Conclusion

These findings indicate that the omentum-wrapped scaffold is capable of enhancing axonal regeneration and functional recovery, which might be served as a potent alternative to nerve autografts. The beneficial effect of omentum-wrapping on nerve regeneration might be related with the proteins produced by omentum.     

COMP-angiopoietin-1 recovers molecular biomarkers of neuropathy and improves vascularisation in sciatic nerve of ob/ob mice

Background

Leptin-deficient ob/ob mice are a model of type 2 diabetes induced peripheral neuropathy. Ob/ob mice exhibit obesity, insulin resistance, hyperglycaemia, and alterations of peripheral nerve fibres and endoneural microvessels. Here we test the hypothesis that cartilage oligomeric matrix protein (COMP)-Ang-1, a soluble and stabile form of Ang-1 which promotes angiogenesis and nerve growth, improves regeneration of nerve fibres and endoneural microvessels in ob/ob mice.

Methods and Findings

COMP-Ang-1 (100 ng/ml) or NaCl were intraperitoneally (i.p.) injected into male (N = 184), 3-month old, ob/ob or ob/+ mice for 7 and 21 days. We measured expression of Nf68, GAP43, Cx32, Cx26, Cx43, and TNFα in sciatic nerves using Western blot analysis. To investigate the inflammation in sciatic nerves, numbers of macrophages and T-cells were counted after immunofluorescence staining. In ultrathin section, number of myelinated/non-mylinated nerve fibers, g-ratio, the thickness of Schwann cell basal lamina and microvessel endothelium were investigated.Endoneural microvessels were reconstructed with intracardial FITC injection. Treatment with COMP-Ang-1 over 21 days significantly reduced fasting blood glucose and plasma cholesterol concentrations compared to saline treated ob/ob mice. In addition, COMP-Ang-1 treatment: 1) up-regulated expression of Nf68 and GAP43; 2) improved expression of gap junction proteins including connexin 32 and 26; 3) suppressed the expression of TNFα and Cx43 and 4) led to decreased macrophage and T-cell infiltration in sciatic nerve of ob/ob mice. The significant changes of sciatic nerve ultrastructure were not observed after 21-day long COMP-Ang-1 treatment. COMP-Ang-1 treated ob/ob mice displayed regeneration of small-diameter endoneural microvessels. Effects of COMP-Ang-1 corresponded to increased phosphorylation of Akt and p38 MAPK upon Tie-2 receptor.

Conclusions

COMP-Ang-1 recovers molecular biomarkers of neuropathy, promotes angiogenesis and suppresses inflammation in sciatic nerves of ob/ob mice suggesting COMP-Ang-1 as novel treatment option to improve morphologic and protein expression changes associated with diabetic neuropathy.     

Axonal-Transport-Mediated Gene Transduction in the Interior of Rat Bone

Background

Gene transduction has been considered advantageous for the sustained delivery of proteins to specific target tissues. However, in the case of hard tissues, such as bone, local gene delivery remains problematic owing to anatomical accessibility limitations of the target sites.

Methodology/Principal Findings

Here, we evaluated the feasibility of exogenous gene transduction in the interior of bone via axonal transport following intramuscular administration of a nonviral vector. A high expression level of the transduced gene was achieved in the tibia ipsilateral to the injected tibialis anterior muscle, as well as in the ipsilateral sciatic nerve and dorsal root ganglia. In sciatic transection rats, the gene expression level was significantly lowered in bone.

Conclusions/Significance

These results suggest that axonal transport is critical for gene transduction. Our study may provide a basis for developing therapeutic methods for efficient gene delivery into hard tissues.     

Gadolinium Enhanced 3D Proton Density Driven Equilibrium MR Imaging in the Evaluation of Cisternal Tumor and Associated Structures: Comparison with Balanced Fast-Field-Echo Sequence

Objectives

Although Gadolinium enhanced bFFE is commonly used to evaluate cisternal tumors, banding artifact may interrupt interpretation and adjacent nerve and vessels differentiation is known to be difficult. We analyzed the qualities of Gd enhanced 3D PDDE in the evaluation of cisternal tumors, comparing with bFFE.

Material and Methods

Forty five cisternal tumors (33 schwannoma and 12 meningioma) on both bFFE and PDDE were retrospectively reviewed. For quantitative analysis, contrast ratios of CSF to tumor and tumor to parenchyma (CR_C/T and CR_T/P) on both sequences were compared by paired t-test. For qualitative analysis, the readers gauged the qualities of the two MR sequences with respect to the degree of demarcating cisternal structures (tumor, basilar artery, AICA, trigeminal nerve, facial nerve and vestibulocochlear nerve).

Results

In quantitative analysis, CR_C/T and CR_T/P on 3D PDDE was significantly lower than that of 3D bFFE (p<0.01). In qualitative analysis, basilar artery, AICA, facial nerve and vestibulocochlear nerves were significantly better demarcated on 3D PDDE than on bFFE (p<0.01). The degree of demarcation of tumor on 3D PDDE was not significantly different with that on 3D bFFE (p = 0.13).

Conclusion

Although the contrast between tumor and the surrounding structures are reduced, Gd enhanced 3D PDDE provides better demarcation of cranial nerves and major vessels adjacent to cisternal tumors than Gd enhanced bFFE     

Immunologic Response to Tumor Ablation with Irreversible Electroporation

Background

Irreversible electroporation (IRE) is a promising technique for the focal treatment of pathologic tissues, which involves placing minimally invasive electrodes within the targeted region. However, the knowledge about the therapeutic efficacy and immune reactions in response to IRE remains in its infancy.

Methods

In this work, to detect whether tumor ablation with IRE could trigger the immunologic response, we developed an osteosarcoma rat model and applied IRE directly to ablate the tumor. In the experiment, 118 SD rats were randomized into 4 groups: the control, sham operation, surgical resection, and IRE groups. Another 28 rats without tumor cell implantation served as the normal non-tumor-bearing group. We analyzed the changes in T lymphocyte subsets, sIL-2R and IL-10 levels in the peripheral blood one day before operation, as well as at 1, 3, 7,14 and 21 days after the operation. Moreover, splenocytes were assayed for IFN-γ and IL-4 production using intracellular cytokine staining one day before the operation, as well as at 7 and 21 days after operation.

Results

We found that direct IRE completely ablated the tumor cells. A significant increase in peripheral lymphocytes, especially CD3⁺ and CD4⁺ cells, as well as an increased ratio of CD4⁺/CD8⁺ were detectable 7 days after operation in both the IRE and surgical resection groups. Compared with the surgical resection group, the IRE group exhibited a stronger cellular immune response. The sIL-2R level of the peripheral blood in the IRE group decreased with time and was significantly different from that in the surgical resection group. Moreover, ablation with IRE significantly increased the percentage of IFN-γ-positive splenocytes.

Conclusion

These findings indicated that IRE could not only locally destroy the tumor but also change the status of cellular immunity in osteosarcoma-bearing rats. This provides experimental evidence for the clinical application of IRE in osteosarcoma treatment.     

Conduction Properties Distinguish Unmyelinated Sympathetic Efferent Fibers and Unmyelinated Primary Afferent Fibers in the Monkey

Background

Different classes of unmyelinated nerve fibers appear to exhibit distinct conductive properties. We sought a criterion based on conduction properties for distinguishing sympathetic efferents and unmyelinated, primary afferents in peripheral nerves.

Methodology/Principal Findings

In anesthetized monkey, centrifugal or centripetal recordings were made from single unmyelinated nerve fibers in the peroneal or sural nerve, and electrical stimuli were applied to either the sciatic nerve or the cutaneous nerve endings, respectively. In centrifugal recordings, electrical stimulation at the sympathetic chain and dorsal root was used to determine the fiber''s origin. In centrifugal recordings, sympathetic fibers exhibited absolute speeding of conduction to a single pair of electrical stimuli separated by 50 ms; the second action potential was conducted faster (0.61 0.16%) than the first unconditioned action potential. This was never observed in primary afferents. Following 2 Hz stimulation (3 min), activity-dependent slowing of conduction in the sympathetics (8.6 0.5%) was greater than in one afferent group (6.7 0.5%) but substantially less than in a second afferent group (29.4 1.9%). In centripetal recordings, most mechanically-insensitive fibers also exhibited absolute speeding to twin pulse stimulation. The subset that did not show this absolute speeding was responsive to chemical stimuli (histamine, capsaicin) and likely consists of mechanically-insensitive afferents. During repetitive twin pulse stimulation, mechanosensitive afferents developed speeding, and speeding in sympathetic fibers increased.

Conclusions/Significance

The presence of absolute speeding provides a criterion by which sympathetic efferents can be differentiated from primary afferents. The differences in conduction properties between sympathetics and afferents likely reflect differential expression of voltage-sensitive ion channels.     

Neuroregenerative effects of olfactory ensheathing cells transplanted in a multi-layered conductive nanofibrous conduit in peripheral nerve repair in rats

Background

The purpose of this study was to evaluate the efficacy of a multi-layered conductive nanofibrous hollow conduit in combination with olfactory ensheathing cells (OEC) to promote peripheral nerve regeneration. We aimed to harness both the topographical and electrical cues of the aligned conductive nanofibrous single-walled carbon nanotube/ poly (_L-lactic acid) (SWCNT/PLLA) scaffolds along with the neurotrophic features of OEC in a nerve tissue engineered approach.

Results

We demonstrated that SWCNT/PLLA composite scaffolds support the adhesion, growth, survival and proliferation of OEC. Using microsurgical techniques, the tissue engineered nerve conduits were interposed into an 8 mm gap in sciatic nerve defects in rats. Functional recovery was evaluated using sciatic functional index (SFI) fortnightly after the surgery. Histological analyses including immunohistochemistry for S100 and NF markers along with toluidine blue staining (nerve thickness) and TEM imaging (myelin sheath thickness) of the sections from middle and distal parts of nerve grafts showed an increased regeneration in cell/scaffold group compared with cell-free scaffold and silicone groups. Neural regeneration in cell/scaffold group was very closely similar to autograft group, as deduced from SFI scores and histological assessments.

Conclusions

Our results indicated that the tissue engineered construct made of rolled sheet of SWCNT/PLLA nanofibrous scaffolds and OEC could promote axonal outgrowth and peripheral nerve regeneration suggesting them as a promising alternative in nerve tissue engineering.     

Venous graft-derived cells participate in peripheral nerve regeneration

Background

Based on growing evidence that some adult multipotent cells necessary for tissue regeneration reside in the walls of blood vessels and the clinical success of vein wrapping for functional repair of nerve damage, we hypothesized that the repair of nerves via vein wrapping is mediated by cells migrating from the implanted venous grafts into the nerve bundle.

Methodology/Principal Findings

To test the hypothesis, severed femoral nerves of rats were grafted with venous grafts from animals of the opposite sex. Nerve regeneration was impaired when decellularized or irradiated venous grafts were used in comparison to untreated grafts, supporting the involvement of venous graft-derived cells in peripheral nerve repair. Donor cells bearing Y chromosomes integrated into the area of the host injured nerve and participated in remyelination and nerve regeneration. The regenerated nerve exhibited proper axonal myelination, and expressed neuronal and glial cell markers.

Conclusions/Significance

These novel findings identify the mechanism by which vein wrapping promotes nerve regeneration.     

L-DOPA Neurotoxicity Is Mediated by Up-Regulation of DMT1?IRE Expression

Background

The mechanisms underlying neurotoxicity caused by L-DOPA are not yet completely known. Based on recent findings, we speculated that the increased expression of divalent metal transporter 1 without iron-response element (DMT1−IRE) induced by L-DOPA might play a critical role in the development of L-DOPA neurotoxicity. To test this hypothesis, we investigated the effects of astrocyte-conditioned medium (ACM) and siRNA DMT-IRE on L-DOPA neurotoxicity in cortical neurons.

Methods and Findings

We demonstrated that neurons treated with L-DOPA have a significant dose-dependent decrease in neuronal viability (MTT Assay) and increase in iron content (using a graphite furnace atomic absorption spectrophotometer), DMT1−IRE expression (Western blot analysis) and ferrous iron (55Fe(II)) uptake. Neurons incubated in ACM with or without L-DOPA had no significant differences in their morphology, Hoechst-33342 staining or viability. Also, ACM significantly inhibited the effects of L-DOPA on neuronal iron content as well as DMT1−IRE expression. In addition, we demonstrated that infection of neurons with siRNA DMT-IRE led to a significant decrease in DMT1−IRE expression as well as L-DOPA neurotoxicity.

Conclusion

The up-regulation of DMT1−IRE and the increase in DMT1−IRE-mediated iron influx play a key role in L-DOPA neurotoxicity in cortical neurons.     

Peripherally-derived BDNF promotes regeneration of ascending sensory neurons after spinal cord injury

Background

The blood brain barrier (BBB) and truncated trkB receptor on astrocytes prevent the penetration of brain derived neurotrophic factor (BDNF) applied into the peripheral (PNS) and central nervous system (CNS) thus restrict its application in the treatment of nervous diseases. As BDNF is anterogradely transported by axons, we propose that peripherally derived and/or applied BDNF may act on the regeneration of central axons of ascending sensory neurons.

Methodology/Principal Findings

The present study aimed to test the hypothesis by using conditioning lesion of the sciatic nerve as a model to increase the expression of endogenous BDNF in sensory neurons and by injecting exogenous BDNF into the peripheral nerve or tissues. Here we showed that most of regenerating sensory neurons expressed BDNF and p-CREB but not p75NTR. Conditioning-lesion induced regeneration of ascending sensory neuron and the increase in the number of p-Erk positive and GAP-43 positive neurons was blocked by the injection of the BDNF antiserum in the periphery. Enhanced neurite outgrowth of dorsal root ganglia (DRG) neurons in vitro by conditioning lesion was also inhibited by the neutralization with the BDNF antiserum. The delivery of exogenous BDNF into the sciatic nerve or the footpad significantly increased the number of regenerating DRG neurons and regenerating sensory axons in the injured spinal cord. In a contusion injury model, an injection of BDNF into the footpad promoted recovery of motor functions.

Conclusions/Significance

Our data suggest that endogenous BDNF in DRG and spinal cord is required for the enhanced regeneration of ascending sensory neurons after conditioning lesion of sciatic nerve and peripherally applied BDNF may have therapeutic effects on the spinal cord injury.     

Preliminary evaluation of the sural nerve using 22-MHz ultrasound: a new approach for evaluation of diabetic cutaneous neuropathy

Background

The application of 22-MHz high-frequency ultrasound allows for visualization of the inner part of the sural nerve. The aim of this study was to evaluate the morphological changes of sural nerves in patients with type 2 diabetes mellitus using ultrasound.

Materials and Methods

The thickness/width (T/W) ratio, the cross-sectional area (CSA) of the sural nerves and the maximum thickness (MT) of the nerve fascicles were measured in 100 patients with type 2 diabetes mellitus and 50 healthy volunteers using 22-MHz ultrasound. Receiver operating characteristic (ROC) curves were plotted to determine the optimal cut-off values as well as the sensitivities and specificities. All parameters were significantly different between the subject and control groups. The ROC curves demonstrated that the MT was the most predictive of diabetic cutaneous neuropathy, with an optimal cut-off value of 0.365 mm that yielded a sensitivity of 90.3% and a specificity of 87.7%.

Conclusions

The results of this study suggest that 22-MHz ultrasound may be a valuable tool for evaluating diabetic cutaneous nerve neuropathy.     

Incorporation of Chitosan Microspheres into Collagen-Chitosan Scaffolds for the Controlled Release of Nerve Growth Factor

Background

Artifical nerve scaffold can be used as a promising alternative to autologous nerve grafts to enhance the repair of peripheral nerve defects. However, current nerve scaffolds lack efficient microstructure and neurotrophic support.

Methods

Microsphere–Scaffold composite was developed by incorporating chitosan microspheres loaded with nerve growth factor (NGF–CMSs) into collagen-chitosan scaffolds (CCH) with longitudinally oriented microchannels (NGF–CMSs/CCH). The morphological characterizations, in vitro release kinetics study, neurite outgrowth assay, and bioactivity assay were evaluated. After that, a 15-mm-long sciatic nerve gap in rats was bridged by the NGF–CMSs/CCH, CCH physically absorbed NGF (NGF/CCH), CCH or nerve autograft. 16 weeks after implantation, electrophysiology, fluoro-gold retrograde tracing, and nerve morphometry were performed.

Results

The NGF–CMSs were evenly distributed throughout the longitudinally oriented microchannels of the scaffold. The NGF–CMSs/CCH was capable of sustained release of bioactive NGF within 28 days as compared with others in vitro. In vivo animal study demonstrated that the outcomes of NGF–CMSs/CCH were better than those of NGF/CCH or CCH.

Conclusion

Our findings suggest that incorporation of NGF–CMSs into the CCH may be a promising tool in the repair of peripheral nerve defects.     

High-Resolution Sonography: A New Technique to Detect Nerve Damage in Leprosy

Background

Leprosy is the most common treatable peripheral nerve disorder worldwide with periods of acute neuritis leading to functional impairment of limbs, ulcer formation and stigmatizing deformities. Since the hallmarks of leprosy are nerve enlargement and inflammation, we used high-resolution sonography (US) and color Doppler (CD) imaging to demonstrate nerve enlargement and inflammation.

Methology/Principal Findings

We performed bilateral US of the ulnar (UN), median (MN), lateral popliteal (LP) and posterior tibial (PT) nerves in 20 leprosy patients and compared this with the clinical findings in these patients and with the sonographic findings in 30 healthy Indian controls.The nerves were significantly thicker in the leprosy patients as compared to healthy controls (p<0.0001 for each nerve). The two patients without nerve enlargements did not have a type 1 or type 2 reaction or signs of neuritis. The kappa for clinical palpation and nerve enlargement by sonography was 0.30 for all examined nerves (0.32 for UN, 0.41 for PN and 0.13 for LP). Increased neural vascularity by CD imaging was present in 39 of 152 examined nerves (26%). Increased vascularity was observed in multiple nerves in 6 of 12 patients with type 1 reaction and in 3 of 4 patients with type 2 reaction. Significant correlation was observed between clinical parameters of grade of thickening, sensory loss and muscle weakness and US abnormalities of nerve echotexture, endoneural flow and cross-sectional area (p<0.001).

Conclusions/Significance

We conclude that clinical examination of enlarged nerves in leprosy patients is subjective and inaccurate, whereas sonography provides an objective measure of nerve damage by showing increased vascularity, distorted echotexture and enlargement. This damage is sonographically more extensive and includes more nerves than clinically expected.     

The Semaphorin 3A Inhibitor SM-345431 Accelerates Peripheral Nerve Regeneration and Sensitivity in a Murine Corneal Transplantation Model

Background

Peripheral nerve damage of the cornea is a complication following surgery or infection which may lead to decreased visual function. We examined the efficacy of the semaphorin 3A inhibitor, SM-345431, in promoting regeneration of peripheral nerves in a mouse corneal transplantation model.

Methodology/Principal Findings

P0-Cre/Floxed-EGFP mice which express EGFP in peripheral nerves cells were used as recipients of corneal transplantation with syngeneic wild-type mouse cornea donors. SM-345431 was administered subconjunctivally every 2 days while control mice received vehicle only. Mice were followed for 3 weeks and the length of regenerating nerves was measured by EGFP fluorescence and immunohistochemistry against βIII tubulin. Cornea sensitivity was also measured by the Cochet-Bonnet esthesiometer. CD31 staining was used to determine corneal neovascularization as a possible side effect of SM-345431. Regeneration of βIII tubulin positive peripheral nerves was significantly higher in SM-345431 treated mice compared to control. Furthermore, corneal sensitivity significantly improved in the SM-345431 group by 3 weeks after transplantation. Neovascularization was limited to the peripheral cornea with no difference between SM-345431 group and control.

Conclusions/Significance

Subconjunctival injections of SM-345431 promoted a robust network of regenerating nerves as well as functional recovery of corneal sensation in a mouse keratoplasty model, suggesting a novel therapeutic strategy for treating neurotrophic corneal disease.     

A Combination of Schwann-Cell Grafts and Aerobic Exercise Enhances Sciatic Nerve Regeneration

Background

Despite the regenerative potential of the peripheral nervous system, severe nerve lesions lead to loss of target-organ innervation, making complete functional recovery a challenge. Few studies have given attention to combining different approaches in order to accelerate the regenerative process.

Objective

Test the effectiveness of combining Schwann-cells transplantation into a biodegradable conduit, with treadmill training as a therapeutic strategy to improve the outcome of repair after mouse nerve injury.

Methods

Sciatic nerve transection was performed in adult C57BL/6 mice; the proximal and distal stumps of the nerve were sutured into the conduit. Four groups were analyzed: acellular grafts (DMEM group), Schwann cell grafts (3×10⁵/2 µL; SC group), treadmill training (TMT group), and treadmill training and Schwann cell grafts (TMT + SC group). Locomotor function was assessed weekly by Sciatic Function Index and Global Mobility Test. Animals were anesthetized after eight weeks and dissected for morphological analysis.

Results

Combined therapies improved nerve regeneration, and increased the number of myelinated fibers and myelin area compared to the DMEM group. Motor recovery was accelerated in the TMT + SC group, which showed significantly better values in sciatic function index and in global mobility test than in the other groups. The TMT + SC group showed increased levels of trophic-factor expression compared to DMEM, contributing to the better functional outcome observed in the former group. The number of neurons in L4 segments was significantly higher in the SC and TMT + SC groups when compared to DMEM group. Counts of dorsal root ganglion sensory neurons revealed that TMT group had a significant increased number of neurons compared to DMEM group, while the SC and TMT + SC groups had a slight but not significant increase in the total number of motor neurons.

Conclusion

These data provide evidence that this combination of therapeutic strategies can significantly improve functional and morphological recovery after sciatic injury.     

Dexamethasone as Adjuvant to Bupivacaine Prolongs the Duration of Thermal Antinociception and Prevents Bupivacaine-Induced Rebound Hyperalgesia via Regional Mechanism in a Mouse Sciatic Nerve Block Model

Background

Dexamethasone has been studied as an effective adjuvant to prolong the analgesia duration of local anesthetics in peripheral nerve block. However, the route of action for dexamethasone and its potential neurotoxicity are still unclear.

Methods

A mouse sciatic nerve block model was used. The sciatic nerve was injected with 60ul of combinations of various medications, including dexamethasone and/or bupivacaine. Neurobehavioral changes were observed for 2 days prior to injection, and then continuously for up to 7 days after injection. In addition, the sciatic nerves were harvested at either 2 days or 7 days after injection. Toluidine blue dyeing and immunohistochemistry test were performed to study the short-term and long-term histopathological changes of the sciatic nerves. There were six study groups: normal saline control, bupivacaine (10mg/kg) only, dexamethasone (0.5mg/kg) only, bupivacaine (10mg/kg) combined with low-dose (0.14mg/kg) dexamethasone, bupivacaine (10mg/kg) combined with high-dose (0.5mg/kg) dexamethasone, and bupivacaine (10mg/kg) combined with intramuscular dexamethasone (0.5mg/kg).

Results

High-dose perineural dexamethasone, but not systemic dexamethasone, combined with bupivacaine prolonged the duration of both sensory and motor block of mouse sciatic nerve. There was no significant difference on the onset time of the sciatic nerve block. There was “rebound hyperalgesia” to thermal stimulus after the resolution of plain bupivacaine sciatic nerve block. Interestingly, both low and high dose perineural dexamethasone prevented bupivacaine-induced hyperalgesia. There was an early phase of axon degeneration and Schwann cell response as represented by S-100 expression as well as the percentage of demyelinated axon and nucleus in the plain bupivacaine group compared with the bupivacaine plus dexamethasone groups on post-injection day 2, which resolved on post-injection day 7. Furthermore, we demonstrated that perineural dexamethasone, but not systemic dexamethasone, could prevent axon degeneration and demyelination. There was no significant caspase-dependent apoptosis process in the mouse sciatic nerve among all study groups during our study period.

Conclusions

Perineural, not systemic, dexamethasone added to a clinical concentration of bupivacaine may not only prolong the duration of sensory and motor blockade of sciatic nerve, but also prevent the bupivacaine-induced reversible neurotoxicity and short-term “rebound hyperalgesia” after the resolution of nerve block.