Evaluation of Stem Cell Laden Collagen + Polycaprolactone + Multi-Walled Carbon Nano-Tubes Nano-Neural Scaffold with and Without Insulin Like Growth Factor-I For Sciatic Nerve Regeneration Post Crush Injury in Wistar Rats.

BACKGROUND/AIMS: All body functions are activated, synchronized and controlled by a substantial, complex network, the nervous system. Upon injury, pathophysiology of the nerve injury proceeds through different paths. The axon may undergo a degenerative retraction from the site of injury for a short distance unless the injury is near to the cell body, in which case it continues to the soma and undergoes retrograde neuronal degeneration. Otherwise, the distal section suffers from Wallerian degeneration, which is marked by axonal swelling, spheroids, and cytoskeleton degeneration. The objective of the study was to evaluate the potential of mesenchymal stem cell laden neural scaffold and insulin-like growth factor I (IGF-I) in nerve regeneration following sciatic nerve injury in a rat model. METHODS: The animals were anaesthetized and a cranio-lateral incision over left thigh was made. Sciatic nerve was exposed and crush injury was introduced for 90 seconds using haemostat at second locking position. The muscle and skin were sutured in routine fashion and thus the rat model of sciatic crush injury was prepared. The animal models were equally distributed into 5 different groups namely A, B, C, D and E and treated with phosphate buffer saline (PBS), carbon nanotubes based neural scaffold only, scaffold with IGF-I, stem cell laden scaffold and stem cell laden scaffold with IGF-I respectively. In vitro scaffold testing was performed. The nerve regeneration was assessed based on physico-neuronal, biochemical, histopathological examination, and relative expression of NRP-1, NRP-2 and GAP-43 and scanning electron microscopy. RESULTS: Sciatic nerve injury model with crush injury produced for 90 seconds was standardized and successfully used in this study. All the biochemical parameters were in normal range in all the groups indicating no scaffold related changes. Physico-neuronal, histopathological, relative gene expression and scanning electron microscopy observations revealed appreciable nerve regeneration in groups E and D, followed by C and B. Restricted to no regeneration was observed in group A. CONCLUSION: Carbon nanotubes based scaffold provided electro-conductivity for proper neuronal regeneration while rat bone marrow-derived mesenchymal stem cells were found to induce axonal sprouting, cellular transformation; whereas IGF-I induced stem cell differentiation, myelin synthesis, angiogenesis and muscle differentiation.

Potential Effects of Stem Cells Derived from the Peripheral Nerve and Adipose Tissue after the Nerve Crush Injury in Control and Obese Rats.

Aim of this study is to investigate effects of stem cells derived from the peripheral nerve and adipose tissues following the nerve crush injury in control and obese rats. For this aim, 41 Wistar Albino female rats were separated into eight equal groups; non-obese control (NOC) obese control (OC), non-obese injury (NOH), obese injury (OH), non-obese adipose (NOY), obese adipose (OY), non-obese nerve (NOPS), obese nerve (OPS). At the end of 8 weeks, all experimental animals without control groups were subjected to nerve crush procedure and sciatic nerve or fat stem cell homogenates were injected on the treatment group rats, and then, recovery process has been observed and histopathological, stereological, electrophysiological analyses and bioinformatic evaluation were made on removed sciatic nerves. Stereological results showed that adipose homogenate gave more successful results than peripheral nerve homogenates in the NOY group in comparison to the NOPS group in terms of myelinated axon number. Peripheral nerve homogenate has shown more successful results in the OPS group in comparison to the OY group. The number of unmyelinated axons was increased following treatment with adipose tissue homogenate in NOY and OY groups. In terms of myelin sheath thickness; we detected that treatments by peripheral nerve and especially adipose tissue homogenates lead to increase in the thickness of the axons of the peripheral nerves belong to the control and obese injury groups. All results showed that mesenchymal stem cell treatment by fresh tissue homogenates is successful in peripheral nerve regeneration and fat tissue is a considerable source of the stem cells for clinical applications.

Peptide ligands targeting FGF receptors promote recovery from dorsal root crush injury via AKT/mTOR signaling.

Background: Fibroblast growth factor receptors (FGFRs) are key targets for nerve regeneration and repair. The therapeutic effect of exogenous recombinant FGFs in vivo is limited due to their high molecular weight. Small peptides with low molecular weight, easy diffusion, low immunogenicity, and nontoxic metabolite formation are potential candidates. The present study aimed to develop a novel low-molecular-weight peptide agonist of FGFR to promote nerve injury repair. Methods: Phage display technology was employed to screen peptide ligands targeting FGFR2. The peptide ligand affinity for FGFRs was detected by isothermal titration calorimetry. Structural biology-based computer virtual analysis was used to characterize the interaction between the peptide ligand and FGFR2. The peptide ligand effect on axon growth, regeneration, and behavioral recovery of sensory neurons was determined in the primary culture of sensory neurons and dorsal root ganglia (DRG) explants in vitro and a rat spinal dorsal root injury (DRI) model in vivo. The peptide ligand binding to other membrane receptors was characterized by surface plasmon resonance (SPR) and liquid chromatography-mass spectrometry (LC-MS)/MS. Intracellular signaling pathways primarily affected by the peptide ligand were characterized by phosphoproteomics, and related pathways were verified using specific inhibitors. Results: We identified a novel FGFR-targeting small peptide, CH02, with seven amino acid residues. CH02 activated FGFR signaling through high-affinity binding with the extracellular segment of FGFRs and also had an affinity for several receptor tyrosine kinase (RTK) family members, including VEGFR2. In sensory neurons cultured in vitro, CH02 maintained the survival of neurons and promoted axon growth. Simultaneously, CH02 robustly enhanced nerve regeneration and sensory-motor behavioral recovery after DRI in rats. CH02-induced activation of FGFR signaling promoted nerve regeneration primarily via AKT and ERK signaling downstream of FGFRs. Activation of mTOR downstream of AKT signaling augmented axon growth potential in response to CH02. Conclusion: Our study revealed the significant therapeutic effect of CH02 on strengthening nerve regeneration and suggested a strategy for treating peripheral and central nervous system injuries.

Beneficial effects of bardoxolone methyl, an Nrf2 activator, on crush-related acute kidney injury in rats.

PURPOSE: The purpose of this study was to investigate the effects of bardoxolone methyl (BM), a nuclear factor erythroid 2-related factor 2 (Nrf2) activator, on acute kidney injury in a rat model of crush syndrome model. METHODS: Sixty-four rats were separated equally into eight groups, sham (sterile saline ip), crush, crush + vehicle (DMSO ip), and crush + BM (10 mg/kg ip) (n = 8). All groups were also divided as 3 and 24 h after decompression. Crush injury was induced by 6 h of direct compression to both hind limbs of the rats with blocks weighing 3.6 kg on each side, followed by 3 and 24 h of decompression. Kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), tumor necrotizing factor-α (TNF-α), transforming growth factor-ß1 (TGF-ß1) concentrations, tissue total oxidant status (TOS) and total antioxidant status (TAS) were measured in the kidneys. Serum creatine kinase (CK), blood urea nitrogen (BUN) and creatinine concentrations were also measured. Glomerular and tubular structures were examined histopathologically. Bcl-2 was measured using immunohistochemistry. Apoptosis was assessed using the TUNEL method. RESULTS: BM treatment reduced KIM-1, NGAL, TNF-α, TGF-ß1, TOS concentrations, and increased TAS concentrations in the kidneys 3 and 24 h after decompression. Serum CK, BUN and creatinine concentrations were also reduced with BM. BM treatment decreased apoptosis in crush-related AKI. The Nrf2 activator BM reversed the crush-induced changes in the experimental rats. CONCLUSION: BM treatment prevented the progression of crush-related AKI in rats possibly through its cytoprotective effects of being an antioxidant, anti-inflammatory and anti-apoptotic agent.

Miconazole alleviates peripheral nerve crush injury by mediating a macrophage phenotype change through the NF-κB pathway.

BACKGROUND: Peripheral nerve injury (PNI) causes motor and sensory defects, has strong impact on life quality and still has no effective therapy. Miconazole is one of the most widely used antifungal drugs; the aims of the study were to investigate the effects of miconazole during sciatic nerve regeneration in a mouse model of sciatic nerve crush injury. METHODS: We established peripheral nerve crush model and investigated the effects of miconazole by multiple aspects. We further studied the potential mechanism of action of miconazole by Western blotting, fluorescence immunohistochemistry, and PCR analysis. RESULTS: Miconazole improves the symptoms of crushed nerve by improving inflammatory cell infiltration and demyelinating myelin of sciatic nerve. Affected by miconazole, the proportion of inflammatory M1 macrophages in the distal part of the sciatic nerve was reduced, and the proportion of anti-inflammatory M2 macrophages was increased. Finally, the neuroprotective properties of miconazole may be regulated by the nuclear factor (NF)-κB pathway. CONCLUSIONS: Our data suggest that miconazole can effectively alleviate PNI, and the mechanism involves mediating a phenotype change of M1/ M2 macrophages. Thus, miconazole may represent a potential therapeutic intervention for nerve crush injury.

Carnosine improves functional recovery and structural regeneration after sciatic nerve crush injury in rats.

AIMS: Peripheral nerve injury represents a substantial clinical problem with insufficient or unsatisfactory treatment options. Current researches have extensively focused on the new approaches for the treatment of peripheral nerve injuries. Carnosine is a naturally occurring pleotropic dipeptide and has many biological functions such as antioxidant property. In the present study, we examined the regenerative ability of carnosine after sciatic nerve crush injury using behavioral, biochemical, histological and ultrastructural evaluations. MATERIALS AND METHODS: Seventy-two rats were divided into six groups including control, sham, crush and carnosine (10, 20 and 40 mg/kg) groups. Crush injury in left sciatic nerve was induced by a small haemostatic forceps. Carnosine was administered for 15 consecutive days after induction of crush injury. Sciatic functional index (SFI) was recorded weekly. Histopathological and ultrastructural evaluations were made using light and electron microscopes, respectively. Sciatic nerve tissue malondialdehyde (MDA), superoxide dismutase (SOD) and tumor necrosis factor-alpha (TNF-α) levels were measured. Gastrocnemius muscle weight was determined. KEY FINDINGS: Carnosine at the doses of 20 and 40 mg/kg accelerated SFI recovery. Wallerian degeneration severity and myelinated fibers density, myelin sheath thickness and diameter as well as ultrastructural changes of myelinated axons were improved. It also recovered nerve tissue biochemical (MDA, SOD and TNF-α) changes induced by crush injury. Muscle weight ratio was reached to near normal values. Our results suggest a regenerative effect of carnosine. Inhibition of oxidative stress and inflammatory pathways, along with provocation of myelination and prevention of muscular atrophy might be involved in this effect of carnosine. SIGNIFICANCE: Carnosine treatment might be considered as a therapeutic agent for peripheral nerve regeneration and its functional recovery.

One-pot construction of a twice-condensed pDNA polyplex system for peripheral nerve crush injury therapy.

Non-viral vector gene delivery is generally limited by its potential toxicity problems, poor transfection abilities, serum stability, or relatively complex construction processes of modified polyplexes. Thus, we develop an efficient and stable polyplex system through convenient construction methods. Here, polyethyleneimine (PEI) 1.8 kDa and glutaraldehyde (GA) are used to construct a novel twice-condensed pDNA polyplex system using a one-pot construction method, including pH-responsive C[double bond, length as m-dash]N linkages by which different PEI molecules on one single polyplex can link with each other. In this system, smaller particle sizes, higher zeta potentials and better serum stabilities are achieved without PEGylation or other chemical modifications using lyophobic segments, but via pH-responsive linkages that ensure the escape of nucleic acids. This polyplex system is used to deliver the pDNA of vascular endothelial growth factor (VEGF) whose half-life period in vivo is only around 30 minutes. Compared with polyplexes prepared using PEI 25 kDa, cells and rats treated with twice-condensed VEGF pDNA polyplexes express significantly more VEGF or myelin basic protein (MBP), and this new polyplex system showed fewer adverse effects in vitro and in vivo. In addition, revascularization and neurogenesis are also discovered in the rat sciatic nerve crush injury model.

Chitosan conduit combined with hyaluronic acid prevent sciatic nerve scar in a rat model of peripheral nerve crush injury.

In the present study, the effects of hyaluronic acid (HA) combined with chitosan conduit on peripheral nerve scarring and regeneration were investigated in a rat model of peripheral nerve crush injury. A total of 60 Sprague-Dawley rats were randomly distributed into four groups (15 rats in each group), in which the nerve was either not treated (control group) or treated with chitosan conduit, hyaluronic acid, or chitosan conduit coupled with hyaluronic acid following clamp injury to the sciatic nerve. The surgical sites were evaluated by assessing the sciatic functional index, the degree of scar adhesions, the numbers of myelinated nerve fibers, the average diameter of myelinated nerve fibers and the myelin sheath thickness. Larger epineurial scar thickness was observed in the control groups compared with the treatment groups at 4, 8 and 12 weeks following surgery. There was no significant difference in scar adhesion among the four groups at 4 weeks following surgery. However, animals receiving chitosan coupled with HA demonstrated better neural recovery, as measured by reduced nerve adherence to surrounding tissues, less scar adhesion, increased number of axons, nerve fiber diameter and myelin thickness. In conclusion, the application of chitosan conduit combined with HA, to a certain extent, inhibited sciatic nerve extraneural scaring and adhesion, and promoted neural regeneration and recovery.

A neuroprotective agent that inactivates prodegenerative TrkA and preserves mitochondria.

Axon degeneration is an early event and pathological in neurodegenerative conditions and nerve injuries. To discover agents that suppress neuronal death and axonal degeneration, we performed drug screens on primary rodent neurons and identified the pan-kinase inhibitor foretinib, which potently rescued sympathetic, sensory, and motor wt and SOD1 mutant neurons from trophic factor withdrawal-induced degeneration. By using primary sympathetic neurons grown in mass cultures and Campenot chambers, we show that foretinib protected neurons by suppressing both known degenerative pathways and a new pathway involving unliganded TrkA and transcriptional regulation of the proapoptotic BH3 family members BimEL, Harakiri,and Puma, culminating in preservation of mitochondria in the degenerative setting. Foretinib delayed chemotherapy-induced and Wallerian axonal degeneration in culture by preventing axotomy-induced local energy deficit and preserving mitochondria, and peripheral Wallerian degeneration in vivo. These findings identify a new axon degeneration pathway and a potentially clinically useful therapeutic drug.

Possible role of antioxidative capacity of (-)-epigallocatechin-3-gallate treatment in morphological and neurobehavioral recovery after sciatic nerve crush injury.

OBJECTIVE This study examined the capacity of the major polyphenolic green tea extract (-)-epigallocatechin-3-gallate (EGCG) to suppress oxidative stress and stimulate the recovery and prompt the regeneration of sciatic nerve after crush injury. METHODS Adult male Wistar rats were randomly assigned to one of 4 groups: 1) Naïve, 2) Sham (sham injury, surgical control group), 3) Crush (sciatic nerve crush injury treated with saline), and 4) Crush+EGCG (sciatic nerve crush injury treated with intraperitoneally administered EGCG, 50 mg/kg). All animals were tested for motor and sensory neurobehavioral parameters throughout the study. Sciatic nerve and spinal cord tissues were harvested and processed for morphometric and stereological analysis. For the biochemical assays, the time points were Day 1, Day 7, Day 14, and Day 28 after nerve injury. RESULTS After sciatic nerve crush injury, the EGCG-treated animals (Crush+EGCG group) showed significantly better recovery of foot position and toe spread and 50% greater improvement in motor recovery than the saline-treated animals (Crush group). The Crush+EGCG group displayed an early hopping response at the beginning of the 3rd week postinjury. Animals in the Crush+EGCG group also showed a significant reduction in mechanical allodynia and hyperalgesia latencies and significant improvement in recovery from nociception deficits in both heat withdrawal and tail flick withdrawal latencies compared with the Crush group. In both the Crush+EGCG and Crush groups, quantitative evaluation revealed significant morphological evidence of neuroregeneration according to the following parameters: mean cross-sectional area of axons, myelin thickness in the sciatic nerve (from Week 4 to Week 8), increase of myelin basic protein concentration and gene expression in both the injured sciatic nerve and spinal cord, and fiber diameter to axon diameter ratio and myelin thickness to axon diameter ratio at Week 2 after sciatic nerve injury. However, the axon area remained much smaller in both the Crush+EGCG and Crush groups compared with the Sham and Naïve groups. The number of axons per unit area was significantly decreased in the Crush+EGCG and Crush groups compared with controls. Sciatic nerve injury produced generalized oxidative stress manifested as a significant increase of isoprostanes in the urine and decrease of the total antioxidant capacity (TAC) of the blood from Day 7 until Day 14. EGCG-treated rats showed significantly less increase of isoprostanes than saline-treated animals and also showed full recovery of TAC levels by Day 14 after nerve injury. In spinal cord tissue analysis, EGCG-treated animals showed induced glutathione reductase and suppressed induction of heme oxygenase 1 gene expression compared with nontreated animals. CONCLUSIONS EGCG treatment suppressed the crush-induced production of isoprostanes and stimulated the recovery of the TAC and was associated with remarkable alleviation of motor and sensory impairment and significant histomorphological evidence of neuronal regeneration following sciatic nerve crush injury in rats. The findings of this study suggest that EGCG can be used as an adjunctive therapeutic remedy for nerve injury. However, further investigations are needed to establish the antioxidative mechanism involved in the regenerative process after nerve injury. Only upregulation of glutathione reductase supports the idea that EGCG is acting indirectly via induction of enzymes or transcription factors.

Blockade of ATP P2X7 receptor enhances ischiatic nerve regeneration in mice following a crush injury.

Preventing damage caused by nerve degeneration is a great challenge. There is a growing body of evidence implicating extracellular nucleotides and their P2 receptors in many pathophysiological mechanisms. In this work we aimed to investigate the effects of the administration of Brilliant Blue G (BBG) and Pyridoxalphosphate-6-azophenyl-2', 4'- disulphonic acid (PPADS), P2X7 and P2 non-selective receptor antagonists, respectively, on sciatic nerve regeneration. Four groups of mice that underwent nerve crush lesion were used: two control groups treated with vehicle (saline), a group treated with BBG and a group treated with PPADS during 28days. Gastrocnemius muscle weight was evaluated. For functional evaluation we used the Sciatic Functional Index (SFI) and the horizontal ladder walking test. Nerves, dorsal root ganglia and spinal cords were processed for light and electron microscopy. Antinoceptive effects of BBG and PPADS were evaluated through von Frey E, and the levels of IL-1ß and TNF-α were analyzed by ELISA. BBG promoted an increase in the number of myelinated fibers and on axon, fiber and myelin areas. BBG and PPADS led to an increase of TNF-α and IL-1ß in the nerve on day 1 and PPADS caused a decrease of IL-1ß on day 7. Mechanical allodynia was reversed on day 7 in the groups treated with BBG and PPADS. We concluded that BBG promoted a better morphological regeneration after ischiatic crush injury, but this was not followed by anticipation of functional improvement. In addition, both PPADS and BBG presented anti-inflammatory as well as antinociceptive effects.

Quercetin promotes motor and sensory function recovery following sciatic nerve-crush injury in C57BL/6J mice.

Injuries and diseases that occur in the nervous system are common and have few effective treatments. Previous studies have shown that quercetin has a therapeutic effect on nervous system injuries, but its potential effects on and mechanisms of action related to behavioral recovery and axonal regrowth have not been investigated. Here, we showed that quercetin administration promotes behavioral recovery following sciatic nerve-crush injury in mice. Long-term evaluation showed that mice administered 20 mg·kg-1·day-1 quercetin for 35 days had a greater sensorimotor recovery compared with all other treatment groups. The mechanisms behind these effects were further investigated, and quercetin was found to regulate the expression of genes involved in regeneration and trophic support. Moreover, quercetin increased cyclic adenosine monophosphate expression and downstream pathway activation, which directly leads to neuronal growth activation in peripheral axon regeneration. In addition, quercetin enhanced axon remyelination, motor nerve conduction velocity and plantar muscle function, indicating that the degree of distal portion hypotrophy during the peripheral axon regeneration process was reduced. These results suggest that quercetin accelerates functional recovery by up-regulating neuronal intrinsic growth capacity and postponing distal atrophy. Overall, quercetin triggered multiple effects to promote behavioral recovery following sciatic nerve-crush injury in mice.

The effect of Riluzole on functional recovery of locomotion in the rat sciatic nerve crush model.

PURPOSE: Peripheral nerve injury (PNI) is common disorder that represents more than 3 % of all traumatic injury cases. One type of PNI, sciatic nerve injury, leads to considerable motoneuron dysfunction. Because Riluzole is clinically approved for the treatment of motoneuron disease, we evaluated whether Riluzole treatment could enhance the nerve regeneration process and improve functional outcome after sciatic nerve crush in rats. METHODS: In acute treatment groups, a single dose of Riluzole (6 and 8 mg/kg) was administered intra-peritoneally 15 min after the crush nerve injury. In the chronic treatment groups, animals were treated with Riluzole (4 and 6 mg/kg/d) for 8 days. Sciatic functional index (SFI) was evaluated for 9 weeks after injury. Furthermore, electrophysiological and morphometric evaluations were performed at the 9th week following injury. RESULTS: Acute and chronic administrations of Riluzole immediately after sciatic nerve crush result in significantly delayed regeneration and reduced motor function outcome. CONCLUSIONS: These findings suggest that early administration of even a single dose of Riluzole after sciatic nerve crush injury can delay motor function recovery. This effect may not depend on its anti-nociceptive activity.

Erythropoietin Enhanced Recovery After Traumatic Nerve Injury: Myelination and Localized Effects.

PURPOSE: We previously found that administration of erythropoietin (EPO) shortens the course of recovery after experimental crush injury to the mouse sciatic nerve. The course of recovery was more rapid than would be expected if EPO's effects were caused by axonal regeneration, which raised the question of whether recovery was instead the result of promoting remyelination and/or preserving myelin on injured neurons. This study tested the hypothesis that EPO has a direct and local effect on myelination in vivo and in vitro. METHODS: Animals were treated with EPO after standard calibrated sciatic nerve crush injury; immunohistochemical analysis was performed to assay for myelinated axons. Combined in vitro neuron-Schwann cell co-cultures were performed to assess EPO-mediated effects directly on myelination and putative protective effects against oxidative stress. In vivo local administration of EPO in a fibrin glue carrier was used to demonstrate early local effects of EPO treatment well in advance of possible neuroregenerative effects. RESULTS: Systemic Administration of EPO maintained more in vivo myelinated axons at the site of nerve crush injury. In vitro, EPO treatment promoted myelin formation and protected myelin from the effects of nitric oxide exposure in co-cultures of Schwann cells and dorsal root ganglion neurons. In a novel, surgically applicable local treatment using Food and Drug Administration-approved fibrin glue as a vehicle, EPO was as effective as systemic EPO administration at time points earlier than those explainable using standard models of neuroregeneration. CONCLUSIONS: In nerve crush injury, EPO may be exerting a primary influence on myelin status to promote functional recovery. CLINICAL RELEVANCE: Mixed injury to myelin and axons may allow the opportunity for the repurposing of EPO for use as a myeloprotective agent in which injuries spare a requisite number of axons to allow early functional recovery.