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The effect of erythropoietin and umbilical cord-derived mesenchymal stem cells on nerve regeneration in rats with sciatic nerve injury
Abstract
Objective
We aimed to investigate the effects of umbilical cord-derived mesenchymal stem cells and erythropoietin on nerve regeneration in the sciatic nerve ‘crush injury’ in a rat model.
Methods
Experimental animals were randomly divided into 5 groups: Crush Injury, Sham, Crush Injury + Erythropoietin, Crush Injury + Mesenchymal Stem Cell, Crush Injury + Erythropoietin + Mesenchymal Stem Cell groups. Crush injury made with bulldog clamp. Mesencyhmal stem cells delivered by enjection locally. Erythropoietin administered by intraperitoneally. On the 0th, 14th and 28th days, all groups underwent a sciatic functional index test. On 28th day, sciatic nerves were harvested and histopathological appearance, axon number and axon diameter of the sciatic nerves were evaluated with Oil Red O staining. Immunoreactivity of nerve growth factor, neurofilament-H and caspase-3 were determined by immunofluorescence staining in nerve tissue.
Results
In histopathological examination, axons and nerve bundles exhibiting normal nerve architecture in the Sham group. Crush Injury + Mesenchymal Stem Cell group has similar histological appearance to the Sham group. The number of axons were higher in the Mesenchymal Stem Cell groups compared to the Crush Injury group. Nerve growth factor immunoreactivity intensity was significantly lower in Crush Injury + Mesenchymal Stem Cell group compared to Crush Injury group. Neurofilament-H density was higher in the treatment groups when compared to the Crush Injury group.
Conclusions
In this study, it was found that umbilical cord-derived mesenchymal stem cells and erythropoietin treatments effects positively regeneration of crush injury caused by bulldog clamp in the sciatic nerve of rats.
Perinatal tissues, such as placenta and umbilical cord contain a variety of somatic stem cell types, spanning from the largely used hematopoietic stem and progenitor cells to the most recently described broadly multipotent epithelial and stromal cells. As perinatal derivatives (PnD), several of these cell types and related products provide an interesting regenerative potential for a variety of diseases. Within COST SPRINT Action, we continue our review series, revising and summarizing the modalities of action and proposed medical approaches using PnD products: cells, secretome, extracellular vesicles, and decellularized tissues. Focusing on the brain, bone, skeletal muscle, heart, intestinal, liver, and lung pathologies, we discuss the importance of potency testing in validating PnD therapeutics, and critically evaluate the concept of PnD application in the field of tissue regeneration. Hereby we aim to shed light on the actual therapeutic properties of PnD, with an open eye for future clinical application. This review is part of a quadrinomial series on functional/potency assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer, anti-inflammation, wound healing, angiogenesis, and regeneration.
The Effect of Pulsed Radiofrequency Application on Nerve Healing After Sciatic Nerve Anastomosis in Rats. In this study, we aimed to evaluate the histomorphological and functional effect of Pulsed Radiofrequency (PRF) application on regeneration after experimental nerve damage in rats. Forty Sprague-Dawley male rats were used in the study. Sciatic nerve incision was applied to all rats and then anastomosis was performed. Twenty rats were separated as the control group, and the remaining 20 rats underwent PRF every day at 42oC, for 120 seconds. The groups were divided into two further subgroups to be sacrificed on the 15th and 30th days. Tissue samples were obtained from all groups at 24 hours and 72 hours after the injury. Sections of sciatic nerve samples were stained with hematoxylin-eosin for light microscopic investigation and prepared for evaluation of ultrastructural changes with transmission electron microscopy. In the evaluation of axon numbers and diameters were seen that the 30th-day RF group had an increase compared to the control group. In the electron microscopic examination, it was observed that myelinated and unmyelinated nerve fiber sheaths had borders that are more regular in the RF group, the nucleus structures of schwann cells were better preserved, mitochondrial damage was less, and the extensions of fibroblast and collagen fibers were smoother than the control group. The findings suggested that PRF application has a positive contribution histologically on nerve healing in the early period after full-layer incision nerve injury anastomosis surgery.
Aim:
We aimed to investigate the effects of erythropoietin, acetyl-l-carnitine, and their combination on nerve regeneration in experimental peripheral nerve injury.
Methods:
Rats were randomly divided into five groups - sham-operated (S), sciatic nerve crush injury (C), C + acetyl-l-carnitine (ALCAR), C + erythropoietin (EPO), and C + EPO + ALCAR. ALCAR (50 mg/kg/day) was administered intraperitoneally, and EPO (5000 U/kg) was injected subcutaneously for 10 days. Functional recovery was evaluated using walking track analysis (sciatic functional index [SFI]), somatosensory evoked potentials (SEPs), thiobarbituric acid reactive substance (TBARS) assay, and caspase-3 and S100 immunoreactivities.
Results:
In SFI analyses, delayed functional recovery was observed in the C group, whereas the functional recovery of rats treated with EPO and ALCAR significantly improved. The latencies of the SEP components were significantly prolonged in C group. In the treatment groups (C + EPO, C + ALCAR, and C + EPO + ALCAR), all recorded values of SEP components significantly decreased. TBARS levels in C group were significantly higher than those in the S group. EPO and ALCAR administration significantly decreased TBARS levels. Caspase-3 immunoreactivity was increased in the C group, whereas it was decreased in the treatment groups. S100 immunolabelling was significantly decreased in the C group. EPO and ALCAR administration caused an increase in the amount of S100-positive cells in all treatment groups.
Conclusion:
EPO and ALCAR administration could accelerate sciatic nerve repair by reducing apoptosis and lipid peroxidation and promoting myelinization. Although both EPO and ALCAR had positive effects on nerve healing, their combined efficacy had no statistically significant effect on peripheral nerve regeneration.
Peripheral nerve injuries (PNIs) are some of the most common types of traumatic lesions affecting the nervous system. Although the peripheral nervous system has a higher regenerative ability than the central nervous system, delayed treatment is associated with disturbances in both distal sensory and functional abilities. Over the past decades, adult stem cell-based therapies for peripheral nerve injuries have drawn attention from researchers. This is because various stem cells can promote regeneration after peripheral nerve injuries by differentiating into neural-line cells, secreting various neurotrophic factors, and regulating the activity of in situ Schwann cells (SCs). This article reviewed research from the past 10 years on the role of stem cells in the repair of PNIs. We concluded that adult stem cell-based therapies promote the regeneration of PNI in various ways.
The remarkable plasticity of Schwann cells allows them to adopt the Remak (non-myelin) and myelin phenotypes, which are specialized to meet the needs of small and large diameter axons, and differ markedly from each other. It also enables Schwann cells initially to mount a strikingly adaptive response to nerve injury and to promote regeneration by converting to a repair-promoting phenotype. These repair cells activate a sequence of supportive functions that engineer myelin clearance, prevent neuronal death, and help axon growth and guidance. Eventually, this response runs out of steam, however, because in the long run the phenotype of repair cells is unstable and their survival is compromised. The re-programming of Remak and myelin cells to repair cells, together with the injury-induced switch of peripheral neurons to a growth mode, gives peripheral nerves their strong regenerative potential. But it remains a challenge to harness this potential and devise effective treatments that maintain the initial repair capacity of peripheral nerves for the extended periods typically required for nerve repair in humans.
The effects of low-level laser therapy (LLLT) and natural latex protein (F1, Hevea brasiliensis) were evaluated on crush-type injuries (15kg) to the sciatic nerve in the expressions of nerve growth factor (NGF) and vascular endothelium growth factor (VEGF) and ultrastructural morphology to associate with previous morphometric data using the same protocol of injury and treatment. Thirty-six male rats were allocated into six experimental groups (n = 6): 1-Control; 2-Exposed nerve; 3-Injured nerve; 4-LLLT (15J/cm2, 780nm, 30mW, Continuous Wave) treated injured nerve; 5-F1 (0,1mg) treated injured nerve; and 6-LLLT&F1 treated injured nerve. Four or eight weeks after, sciatic nerve samples were processed for analysis. NGF expression were higher (p
After peripheral nerve crush injury, the fibers of distal nerve segments gradually disintegrate, and axons regrow from the proximal nerve segment, eventually reaching the target organ. However, the axon regeneration is generally not sufficient for the recovery of neurological function, so drug therapy is necessary. In the current study, we explored the effect of Tanshinone IIA in nerve regeneration in a sciatic nerve crush injury model using Sprague Dawley rats. The rats were administered 45 mg/kg of Tanshinone IIA once daily. Motor behavior and tibialis anterior muscle mass were assessed, and histological analysis of the sciatic nerve and lumbar spinal cord were conducted. The results showed that the administration of Tanshinone IIA improved nerve growth and motor function, and resulted in a marked decrease of neuronal death. The findings of this exploratory study suggest that Tanshinone IIA alleviates injury and boosts regeneration after nerve crush injury in a rat model of sciatic nerve injury.
Background and objectives:
Mesenchymal stem cells (MSCs) are self-renewing, non-specialized cells used clinically in tissue regeneration and sourced from bone marrow, peripheral blood, umbilical cord blood and umbilical cord tissue (UCT). To demonstrate an alternative method for MSC detection, cryopreserved UCT and expanded MSC were screened for MSC markers CD73, CD90, CD105 and CDH-11 by RT-PCR.
Methods and results:
Human UCT were washed, sectioned, cryopreserved with 10% DMSO and stored in the vapor phase of liquid nitrogen. Fresh and frozen UCT samples were expanded for MSC. UCT and MSC were processed for RNA and screened for CD73, CD90, CD105 and CDH-11 mRNA by RT-PCR. CD73, CD90 and CD105 were detected by flow cytometry and CDH-11 was detected by Western blotting. Short and long-term frozen UCT shows a loss of mRNA expression for CD73 at 33.2±34.0%, CD90 at 6.2±8.2%, CD105 at 17.7±21.6% and CDH-11 at 30.1±26.7% but was not statistically significant to indicate the deterioration. Expanded MSCs from fresh UCT expressed 0.09±0.07-fold CD73, 0.17±0.11-fold CD90, 0.04±0.06-fold CD105 and 0.14±0.08-fold CDH-11. Expanded MSCs from frozen UCTs expressed 0.09±0.06-fold CD73, 0.13±0.06-fold CD90, 0.04±0.05-fold CD105 and 0.11±0.06-fold CDH-11 and confirmed by flow cytometry and Western blotting.
Conclusion:
CD73, CD90, CD105 and CDH-11 were detected by RT-PCR in cryopreserved UCT and MSC expansion. CDH-11 appears as a useful single target MSC marker for quick screening.
Purpose:
The aim of this study was to define a sutureless peripheral nerve repair technique with a vein graft and bone marrow-derived stem cells (BMSC) and compare it to epineural repair.
Materials and methods:
Thirty Wistar Albino rats were divided into five groups evenly. In the control group (C), epineural repair was performed. In the SV (suture + vein) and MSV (BMSC + suture + vein) groups, epineural repair was wrapped with a vein graft. In the V (vein) and MV (vein + BMSC) groups, sutureless repair using a vein graft was performed by taking sutures away from the regeneration site. Rats were evaluated with pinprick, toe spread tests and sciatic nerve index (SFI) at 4th, 8th, and 12th weeks. They were sacrificed at 12th week, repair sites were harvested and evaluated immunohistochemically.
Results:
There was no difference in pinprick and toe spread tests between the groups at 12th week. The mean SFI was -76.5 ± 3.7, -65.2 ± 11.7, -46.2 ± 19.4, -68.8 ± 9.8, -56 ± 8.8 in the C, SV, MSV, V, MV groups, respectively. The MSV group showed significantly the best SFI results (P < .05). NF-H immunostaining scores were as C; 1 ± 0.18, SV; 2.5 ± 0.36, MSV; 4 ± 0.49, V; 1.56 ± 0.54, MV; 3 ± 0.39, whereas GAP-43 scores were as C; 1 ± 0.31, SV; 2.66 ± 0.56, MSV; 4.50 ± 0.23, V; 2 ± 0.23, MV; 3 ± 0.6. The best nerve regeneration according to immunostaining results was observed in the MSV group (P < .05). The mean fibrosis area was 221.5 ± 25.9, 101.6 ± 7.1, 121.3 ± 18.8, 150.3 ± 12.1, 152.4 ± 11.8 μm2in the above groups, respectively. SV and MSV groups showed the significantly less fibrosis area (P < .05).
Conclusion:
Epineural suture repair combined with vein wrapping and BMSCs (MSV) showed the best SFI, GAP-43, and NF-H immunostaining results.
Studies have confirmed that bone marrow-derived mesenchymal stem cells (MSCs) can be used for treatment of several nervous system diseases. However, isolation of bone marrow-derived MSCs (BMSCs) is an invasive and painful process and the yield is very low. Therefore, there is a need to search for other alterative stem cell sources. Adipose-derived MSCs (ADSCs) have phenotypic and gene expression profiles similar to those of BMSCs. The production of ADSCs is greater than that of BMSCs, and ADSCs proliferate faster than BMSCs. To compare the effects of venous grafts containing BMSCs or ADSCs on sciatic nerve injury, in this study, rats were randomly divided into four groups: sham (only sciatic nerve exposed), Matrigel (MG; sciatic nerve injury + intravenous transplantation of MG vehicle), ADSCs (sciatic nerve injury + intravenous MG containing ADSCs), and BMSCs (sciatic nerve injury + intravenous MG containing BMSCs) groups. Sciatic functional index was calculated to evaluate the function of injured sciatic nerve. Morphologic characteristics of nerves distal to the lesion were observed by toluidine blue staining. Spinal motor neurons labeled with Fluoro-Gold were quantitatively assessed. Compared with sham-operated rats, sciatic functional index was lower, the density of small-diameter fibers was significantly increased, and the number of motor neurons significantly decreased in rats with sciatic nerve injury. Neither ADSCs nor BMSCs significantly improved the sciatic nerve function of rats with sciatic nerve injury, increased fiber density, fiber diameters, axonal diameters, myelin sheath thickness, and G ratios (axonal diameter/fiber diameter ratios) in the sciatic nerve distal to the lesion site. There was no significant difference in the number of spinal motor neurons among ADSCs, BMSCs and MG groups. These results suggest that neither BMSCs nor ADSCs provide satisfactory results for peripheral nerve repair when using MG as the conductor for engraftment.
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.
Most type 2 diabetic patients are obese who have increased number of visceral adipocytes. Those visceral adipocytes release several factors that enhance insulin resistance making diabetic treatment ineffective. It is known that significant percentages of visceral adipocytes are derived from mesenchymal stem cells and high glucose enhances adipogenic differentiation of mouse bone marrow-derived MSCs (BM-MSCs). However, the effect of high glucose on adipogenic differentiation of human bone marrow and gestational tissue-derived MSCs is still poorly characterized. This study aims to investigate the effects of high glucose on proliferation as well as adipogenic and osteogenic differentiation of human MSCs derived from bone marrow and several gestational tissues including chorion, placenta, and umbilical cord. We found that high glucose reduced proliferation but enhanced adipogenic differentiation of all MSCs examined. The expression levels of some adipogenic genes were also upregulated when MSCs were cultured in high glucose. Although high glucose transiently downregulated the expression levels of some osteogenic genes examined, its effect on the osteogenic differentiation levels of the MSCs is not clearly demonstrated. The knowledge gained from this study will increase our understanding about the effect of high glucose on adipogenic differentiation of MSCs and might lead to an improvement in the diabetic treatment in the future.
Unlike other tissues in the body, peripheral nerve regeneration is slow and usually incomplete. Less than half of patients who undergo nerve repair after injury regain good to excellent motor or sensory function and current surgical techniques are similar to those described by Sunderland more than 60 years ago. Our increasing knowledge about nerve physiology and regeneration far outweighs our surgical abilities to reconstruct damaged nerves and successfully regenerate motor and sensory function. It is technically possible to reconstruct nerves at the fascicular level but not at the level of individual axons. Recent surgical options including nerve transfers demonstrate promise in improving outcomes for proximal nerve injuries and experimental molecular and bioengineering strategies are being developed to overcome biological roadblocks limiting patient recovery.
Peripheral nerve injuries and neuropathies lead to profound functional deficits. Here, we have demonstrated that muscle-derived stem/progenitor cells (MDSPCs) isolated from adult human skeletal muscle (hMDSPCs) can adopt neuronal and glial phenotypes in vitro and ameliorate a critical-sized sciatic nerve injury and its associated defects in a murine model. Transplanted hMDSPCs surrounded the axonal growth cone, while hMDSPCs infiltrating the regenerating nerve differentiated into myelinating Schwann cells. Engraftment of hMDSPCs into the area of the damaged nerve promoted axonal regeneration, which led to functional recovery as measured by sustained gait improvement. Furthermore, no adverse effects were observed in these animals up to 18 months after transplantation. Following hMDSPC therapy, gastrocnemius muscles from mice exhibited substantially less muscle atrophy, an increase in muscle mass after denervation, and reorganization of motor endplates at the postsynaptic sites compared with those from PBS-treated mice. Evaluation of nerve defects in animals transplanted with vehicle-only or myoblast-like cells did not reveal histological or functional recovery. These data demonstrate the efficacy of hMDSPC-based therapy for peripheral nerve injury and suggest that hMDSPC transplantation has potential to be translated for use in human neuropathies.
Intravital microscopy has become increasingly popular over the past few decades because it provides high-resolution and real-time information about complex biological processes. Technological advances that allow deeper penetration in live tissues, such as the development of confocal and two-photon microscopy, together with the generation of ever-new fluorophores that facilitate bright labelling of cells and tissue components have made imaging of vertebrate model organisms efficient and highly informative. Genetic manipulation leading to expression of fluorescent proteins is undoubtedly the labelling method of choice and has been used to visualize several cell types in vivo. This approach, however, can be technically challenging and time consuming. Over the years, several dyes have been developed to allow rapid, effective and bright ex vivo labelling of cells for subsequent transplantation and imaging. Here, we review and discuss the advantages and limitations of a number of strategies commonly used to label and track cells at high resolution in vivo in mouse and zebrafish, using fluorescence microscopy. While the quest for the perfect label is far from achieved, current reagents are valuable tools enabling the progress of biological discovery, so long as they are selected and used appropriately.
Nerve tissue presents inherent difficulties for its effective regeneration. Stem cell transplantation is considered an auspicious treatment for neuronal injuries. Recently, human dental mesenchymal stem cells (DMSCs) have received extensive attention in the field of regenerative medicine due to their accessibility and multipotency. Since their origin is within the neural crest, they can be differentiated into neural crest-derived cells including neuron and glia cells both in vitro and in vivo. DMSCs are also able to secrete a wide variety of neurotrophins and chemokines, which promote neuronal cells to survival and differentiation. Experimental evidence has shown that human DMSCs engraftment recovered neuronal tissue damage in animal models of central nervous system injuries. Human DMSCs can be a new hope for treatment of nervous system diseases and deficits such as spinal cord injury, stroke and Parkinson's disease.
Background
The physiological function of p38α, which is an isoform of p38 MAPK, has been investigated previously in several studies using pharmacological inhibitors. However, the results regarding whether p38α promotes or inhibits nerve regeneration in vivo have been controversial.
Methods
We generated novel p38α mutant mice (sem mice) with a point mutation in the region encoding the p38α substrate-docking-site, which serves as a limited loss-of-function model of p38α. In the present study, we utilized sem mice and wild-type littermates (wt mice) to investigate the physiological role of p38α in nerve regeneration following crush injuries.
Results
At four weeks after crush injury, the average axon diameter and the average axon area in sem mice were significantly smaller than those in wt mice. The average myelin sheath thickness in sem mice was reduced compared to wt mice, but no significant difference was observed in the G-ratio between the two groups. The sciatic functional index value demonstrated that functional nerve recovery in sem mice following crush injury was delayed, which is consistent with the histological findings. To investigate the underlying mechanisms of these findings, we examined inflammatory responses of the sciatic nerve by immunohistochemistry and western blotting. At an early phase following crush injury, sem mice showed remarkably lower expression of inflammatory cytokines, such as TNF-α and IL-1β, than wt mice. The expression of Caspase-3 and Tenascin-C were also lower in sem mice. Conversely, at a late phase of the response, sem mice showed considerably higher expression of TNF-α and of IL-1β with lower expression of S-100 than wt mice.
Conclusions
This is the first study of the physiological role of p38 MAPK in nerve regeneration that does not rely on the use of pharmacological inhibitors. Our results indicate that p38α insufficiency may cause an inflammatory disorder, resulting in a delay of histological and functional nerve recovery following crush injury. We conclude that p38 MAPK has an important physiological role in nerve regeneration and may be important for controlling both initiation of inflammation and recovery from nerve injury.
Determining how normal and leukemic stem cells behave in vivo, in a dynamic and noninvasive way, remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes, which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context, we evaluated the possibility to use gene transfer-free labeling technologies. The lipophilic dye 3,3,3',3' tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate [DiI], DiD, DiR, and PKH26) can give rise to microenvironmental contamination, including when used in suboptimal concentration, after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually, we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non-genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches.
The tissue protective functions of the hematopoietic growth factor erythropoietin (EPO) are independent of its action on erythropoiesis. EPO and its receptors (EPOR) are expressed in multiple brain cells during brain development and upregulated in the adult brain after injury. Peripherally administered EPO crosses the blood-brain barrier and activates in the brain anti-apoptotic, anti-oxidant and anti-inflammatory signaling in neurons, glial and cerebrovascular endothelial cells and stimulates angiogenesis and neurogenesis. These mechanisms underlie its potent tissue protective effects in experimental models of stroke, cerebral hemorrhage, traumatic brain injury, neuroinflammatory and neurodegenerative disease. The preclinical data in support of the use of EPO in brain disease have already been translated to first clinical pilot studies with encouraging results with the use of EPO as a neuroprotective agent.
Executive functions, learning and attention are imperative facets of cognitive performance, affected in many neuropsychiatric disorders. Recently, we have shown that recombinant human erythropoietin improves cognitive functions in patients with chronic schizophrenia, and that it leads in healthy mice to enhanced hippocampal long-term potentiation, an electrophysiological correlate of learning and memory. To create an experimental basis for further mechanistic insight into erythropoietin-modulated cognitive processes, we employed the Five Choice Serial Reaction Time Task. This procedure allows the study of the effects of erythropoietin on discrete processes of learning and attention in a sequential fashion.
Male mice were treated for 3 weeks with erythropoietin (5,000 IU/kg) versus placebo intraperitoneally every other day, beginning at postnatal day 28. After termination of treatment, mice were started on the Five Choice Serial Reaction Time Task, with daily training and testing extending to about 3 months.Overall, a significantly higher proportion of erythropoietin-treated mice finished the task, that is, reached the criteria of adequately reacting to a 1.0 sec flash light out of five arbitrarily appearing choices. During acquisition of this capability, that is, over almost all sequential training phases, learning readouts (magazine training, operant and discriminant learning, stability of performance) were superior in erythropoietin-treated versus control mice.
Early erythropoietin treatment leads to lasting improvement of cognitive performance in healthy mice. This finding should be exploited in novel treatment strategies for brain diseases.
The function of neurofilaments, the major component in large myelinated neurons, is not well understood even though they were discovered as structures over 100 years ago. Recent studies have suggested that neuro-filaments are closely related to many neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson disease Alzheimer disease, and diabetes. Using in vitro assays, cultures and transgenic mice, these studies provided new insights into neurofilament function. The function of each subunit, the relationship of neurofilaments with other cytoskeletal elements and their clinical significance are topics of increasing attention.
The umbilical cord contains an inexhaustible, noncontroversial source of stem cells for therapy. In the U.S., stem cells found in the umbilical cord are routinely placed into bio-hazardous waste after birth. Here, stem cells derived from human umbilical cord Wharton's Jelly, called umbilical cord matrix stem (UCMS) cells, are characterized. UCMS cells have several properties that make them of interest as a source of cells for therapeutic use. For example, they 1) can be isolated in large numbers, 2) are negative for CD34 and CD45, 3) grow robustly and can be frozen/thawed, 4) can be clonally expanded, and 5) can easily be engineered to express exogenous proteins. UCMS cells have genetic and surface markers of mesenchymal stem cells (positive for CD10, CD13, CD29, CD44, and CD90 and negative for CD14, CD33, CD56, CD31, CD34, CD45, and HLA-DR) and appear to be stable in terms of their surface marker expression in early passage (passages 4-8). Unlike traditional mesenchymal stem cells derived from adult bone marrow stromal cells, small populations of UCMS cells express endoglin (SH2, CD105) and CD49e at passage 8. UCMS cells express growth factors and angiogenic factors, suggesting that they may be used to treat neurodegenerative disease. To test the therapeutic value of UCMS cells, undifferentiated human UCMS cells were transplanted into the brains of hemiparkinsonian rats that were not immune-suppressed. UCMS cells ameliorated apomorphine-induced rotations in the pilot test. UCMS cells transplanted into normal rats did not produce brain tumors, rotational behavior, or a frank host immune rejection response. In summary, the umbilical cord matrix appears to be a rich, noncontroversial, and inexhaustible source of primitive mesenchymal stem cells.
BACKGROUND: Recovery of motor and sensory function from peripheral nerve injury is relatively slow and incomplete. It is a difficult problem for orthopedic surgeons that mainly leads to the decline in the quality of life in patients. OBJECTIVE: To conclude the methods and corresponding outcomes in peripheral nerve regeneration by analyzing the new treatment means for peripheral nerve injury. METHODS: PubMed, Wanfang, CNKI databases were retrieved for relevant articles using key words of “nerve injury, regeneration”, and then retrieval data were sorted and analyzed. RESULTS AND CONCLUSION: In recent years, in-depth studies on peripheral nerve repair have been made in the following aspects: surgical mode, drug, cytokine, gene transfer and biomaterials as well as traditional Chinese medicine. If the detect size is four times longer than the diameter of nerves, the nerve regeneration chamber can achieve good outcomes. The methods of restoring nerve continuity following nerve injury are developed from surgical anastomosis to photochemohistological method, thermal laser welding, plastic repair and other emerging technologies. Studies have found that plasminogen activator, nerve growth factor, neurotrophic factor, recombinant erythropoietin, human tissue kallikrein, B vitamins and their derivatives, herbal preparations, immunosuppressive agents all can promote nerve regeneration. © 2016, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.
Human mesenchymal stem cells (MSCs) are a heterogeneous subset of nonhematopoietic multipotent stromal stem cells and can differentiate into mesodermal lineage, such as adipocytes, osteocytes, and chondrocytes, as well as ectodermal and endodermal lineages. Human umbilical cord (UC) is one of the most promising sources of MSCs. However, the molecular and cellular characteristics of UC-derived MSCs (UC-MSCs) require extensive investigations, which are hampered by the limited lifespan and the diminished potency over passages. Here, we used the piggyBac transposon-based simian virus 40 T antigen (SV40T) immortalization system and effectively immortalized UC-MSCs, yielding the iUC-MSCs. A vast majority of the immortalized lines are positive for MSC markers but not for hematopoietic markers. The immortalization phenotype of the iUC-MSCs can be effectively reversed by flippase recombinase-induced the removal of SV40T antigen. While possessing long-term proliferation capability, the iUC-MSCs are not tumorigenic in vivo. Upon bone morphogenetic protein 9 (BMP9) stimulation, the iUC-MSC cells effectively differentiate into osteogenic, chondrogenic, and adipogenic lineages both in vitro and in vivo, which is indistinguishable from that of primary UC-MSCs, indicating that the immortalized UC-MSCs possess the characteristics similar to that of their primary counterparts and retain trilineage differentiation potential upon BMP9 stimulation. Therefore, the engineered iUC-MSCs should be a valuable alternative cell source for studying UC-MSC biology and their potential utilities in immunotherapies and regenerative medicine.
Injury to the brain and spinal cord has devastating consequences because adult central nervous system (CNS) axons fail to regenerate. Injury to the peripheral nervous system (PNS) has a better prognosis, because adult PNS neurons support robust axon regeneration over long distances. CNS axons have some regenerative capacity during development, but this is lost with maturity. Two reasons for the failure of CNS regeneration are extrinsic inhibitory molecules, and a weak intrinsic capacity for growth. Extrinsic inhibitory molecules have been well characterized, but less is known about the neuron‐intrinsic mechanisms which prevent axon re‐growth. Key signaling pathways and genetic/epigenetic factors have been identified which can enhance regenerative capacity, but the precise cellular mechanisms mediating their actions have not been characterized. Recent studies suggest that an important prerequisite for regeneration is an efficient supply of growth‐promoting machinery to the axon; however, this appears to be lacking from non‐regenerative axons in the adult CNS. In the first part of this review, we summarize the evidence linking axon transport to axon regeneration. We discuss the developmental decline in axon regeneration capacity in the CNS, and comment on how this is paralleled by a similar decline in the selective axonal transport of regeneration‐associated receptors such as integrins and growth factor receptors. In the second part, we discuss the mechanisms regulating selective polarized transport within neurons, how these relate to the intrinsic control of axon regeneration, and whether they can be targeted to enhance regenerative capacity. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 2018
Several studies have demonstrated extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) could promote neural regeneration following central nervous system injury. However, the therapeutic effects of MSC-EVs in peripheral nerve regeneration remain poorly understood. This study was aimed to investigate the therapeutic effects of local application of rat bone marrow mesenchymal stem cell (BMSCs) derived EVs in rat sciatic nerve crush injury model. EVs were isolated from the culture supernatants of BMSCs under serum-free conditioned medium, and identified by transmission electron microscopy (TEM) and scanning electron microscope (SEM). Then, the crush-injured segment of rat was treated by BMSC-EVs or PBS, and assessed the nerve regeneration. Results showed that BMSC-EVs size ranged from 40 to 300 nm and had a typical membrane structure under TEM and SEM. BMSC-EVs treatment promote the function recovery by sciatic function Index, improve the histomorphometric repair in nerve regeneration, and increase the expression of GAP-43, a marker for axon regeneration. The present study demonstrated that BMSC-EVs could promote the functional recovery and nerve regeneration of crush-injured sciatic nerves in rat. BMSC-EVs provide a novel cell-free therapeutic approach to peripheral nerve therapy.
Introduction:
Erythropoietin (EPO) has been identified as a neuroregenerative agent. We hypothesize that it may accelerate recovery after crush injury and may vary with crush severity.
Methods:
Mice were randomized to mild, moderate, or severe crush of the sciatic nerve and were treated with EPO or vehicle control after injury. The sciatic function index (SFI) was monitored over the first week. Microstructural changes were analyzed by immunofluorescence for neurofilament (NF) and myelin (P0 ), and electron microscopy was used to assess ultrastructural changes.
Results:
In moderate crush injuries, EPO significantly improved SFI at 7 days post-injury, an effect not observed with other severity levels. Increases in the ratio of P0 to NF were observed after EPO treatment in moderate crush injuries. Electron microscopy demonstrated endothelial cell hypertrophy in the EPO group.
Conclusions:
EPO accelerates recovery in moderately crushed nerves, which may be through effects on myelination and vascularization. Injury severity may influence the efficacy of EPO. Muscle Nerve, 2016.
It has been proposed that depression is associated with dysfunction of hippocampal plasticity. Novel hypotheses suggest that antidepressants induce neuronal structural plasticity, although the underlying mechanisms still remain unclear. Therefore, the aim of this study was to investigate the effects of amitriptyline on levels of phosphorylated heavy neurofilament subunit (NF-H) in the hippocampus of mice exposed to acute and chronic behavioral despair paradigms. Immunoblotting experiments showed that animals exposed to the tail suspension test (TST) displayed diminished levels of pNF-H 24 h after testing. Repeated administration of amitriptyline (10 mg/kg i.p.) prevented this decreased hippocampal phosphorylation of NF-H. Conversely, administration of citalopram (10 mg/kg i.p.) left unchanged pNF-H levels. The expression of pNF-H was also analyzed by immunofluorescence in mice exposed to the unpredictable chronic mild stress paradigm (UCMS), an experimental model of depression. Mice that developed a depressive-like behavior showed a decreased pNF-H immunostaining selectively in the hippocampal CA3 region. Chronic administration of amitriptyline reversed the despaired behavior induced by exposure to UCMS paradigm and, fully recovered pNF-H labeling to control values. Our results indicate that the cytoskeletal remodeling induced by amitriptyline in the hippocampal CA3 region might underpin its behavioral efficacy. Hippocampal alterations of the NF appeared associated with the mechanism of this antidepressant drug and may contribute to its psychotherapeutic actions.
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.
Although regimens of stem cell implantation can elicit functional recovery following peripheral nerve injury, the degree of outcome is still limited. This study evaluated the synergistic effects of cold-water swimming (CWS) and mesenchymal stem cell (MSC) transplantation on functional recovery of crushed sciatic nerve in rats.
Forty Sprague-Dawley rats that had their sciatic nerve crushed during surgery were randomly divided into four groups: MSCCWS group, treated with combination of MSC and CWS; MSC group, treated with MSC alone; CWS group, treated with CWS alone; and non-treated group, without any treatments. The sciatic function index (SFI), vertical activity (VA), ankle activity (AA) and electrophysiological study were examined before, immediately after surgery, after the treatment and after 4 weeks from treatment. Morphological and S100 immunohistochemical studies were also performed.
The MSCCWS group showed a greater improvement in SFI, VA, AA, peak amplitudes and onset latencies of compound muscle action potential (CMAP) in sciatic nerve and infiltration of immune cells with significant difference from the MSC, CWS and non-treated groups (P < 0.05).
MSC transplantation combined with CWS could achieve better results in functional recovery than a single treatment of MSC alone or CWS alone in nerve crush injury.
Cisplatin is an anticancer drug and it has neurotoxic effects. On the other hand, the neuroprotective effect of selenium was observed in previous studies. However, the effect of selenium on cisplatin-induced neurotoxicity has not been studied yet. Therefore, we aimed to investigate whether selenium prevent cisplatin-induced neurotoxicity. Twenty-one male Wistar albino rats were divided into three groups: Control (C), cisplatin (CS), cisplatin and selenium (CSE, n = 7 in each group). Cisplatin (12 mg/kg/day, i.p.) was administered for 3 days to CS and CSE groups. Also, CSE group received via oral gavage 3 mg/kg/day (twice-a-day as 1.5 mg/kg) selenium 5 days before of cisplatin injection and continued for 11 consecutive days. The same volumes of saline were intraperitoneally and orally administered to C group at same time. At the end of experimental protocol, electrophysiological and histopathological examinations were performed. The nerve conduction velocity, amplitude of compound action potential and number of axon of CS group were significantly lower than the C group. However, the same parameters of CSE group were significantly higher than the CS group. Although, cisplatin has a peripheral neurotoxic effect in rats, this effect was partially prevented by selenium treatment. Thus, it appears that co-administration of selenium and cisplatin may be a useful approach to decrease severity of peripheral neurotoxicity.
Patients with peripheral nerve injuries, especially severe injury, often face poor nerve regeneration and incompletely functional recovery, even after surgical nerve repair. Current researches have extensively focused on the new approaches for the treatment of peripheral nerve injuries. This review summarizes treatments of peripheral nerve injures, from conventional suturing method, to conduit coaptation with stem cell and growth factor, and review the developments of research and clinical application of these therapies.
Bone marrow-derived mesenchymal stem cells (BMSCs) preferentially migrate to the injured tissue but with limited efficiency. Here we investigated the effect of erythropoietin (EPO) treatment on the BMSC migration to the acute kidney injury (AKI) microenvironment. The possible mechanisms were also discussed. A hypoxia/re-oxygenation (HR) model of renal tubular epithelial cells (RTECs) was established to generate AKI in vitro, and a chemotaxis experiment was conducted using the transwell chamber. EPO treatment enhanced the BMSC migration to the HR-RTEC culturing chamber in a SDF-1 level-dependent manner, which was fully inhibited by the treatment of anti-SDF-1 antibody. The BMSC migration could also be partly blocked by LY294002 (phosphoinositide 3-kinase (PI3K) inhibitor) and PD98059 (MAPK inhibitor). Western blot analysis showed that phosphorylated Akt and phosphorylated MAPK in BMSCs were enhanced by EPO treatment. In the in vivo experiment, BMSCs were transplanted into the AKI mice and EPO was subcutaneously injected. The results showed that EPO injection increased the SDF-1 protein expression and BMSC accumulation in the renal tissue, which was consistent with a decent improvement of renal function. In addition, the BMSC accumulation in the renal tissue was blocked by anti-SDF-1 antibody, LY294002 or PD98059. Our data suggest that AKI microenvironment had a directional chemotactic effect on BMSCs, which could be further enhanced by the EPO treatment. The increased SDF-1 level in the AKI microenvironment and the activations of PI3K/AKT and MAPK in BMSCs were the possible mechanisms for the effect of EPO. Therefore, BMSC transplantation combined with EPO injection can be a novel and effective approach for AKI repair.
Cellular systems implanted into an injured nerve may produce growth factors or extracellular matrix molecules, modulate the inflammatory process and eventually improve nerve regeneration. In the present study, we evaluated the therapeutic value of human umbilical cord matrix MSCs (HMSCs) on rat sciatic nerve after axonotmesis injury associated to Vivosorb® membrane. During HMSCs expansion and differentiation in neuroglial-like cells, the culture medium was collected at 48, 72 and 96 h for nuclear magnetic resonance (NMR) analysis in order to evaluate the metabolic profile. To correlate the HMSCs ability to differentiate and survival capacity in the presence of the Vivosorb® membrane, the [Ca2+]i of undifferentiated HMSCs or neuroglial-differentiated HMSCs was determined by the epifluorescence technique using the Fura-2AM probe. The Vivosorb® membrane proved to be adequate and used as scaffold associated with undifferentiated HMSCs or neuroglial-differentiated HMSCs. In vivo testing was carried out in adult rats where a sciatic nerve axonotmesis injury was treated with undifferentiated HMSCs or neuroglial differentiated HMSCs with or without the Vivosorb® membrane. Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index (SFI), extensor postural thrust (EPT), and withdrawal reflex latency (WRL).
The aim of this study was to evaluate nerve regeneration in relation to the transcription factor, Activating Transcription Factor 3 (ATF 3), and an apoptotic marker, caspase 3, in the Schwann cells of diabetic BB rats (i.e. display type 1 diabetes phenotype). Sciatic nerves in healthy Wistar rats and in diabetic BB rats were transected and immediately repaired. Axonal outgrowth (neurofilament staining) and expression of ATF 3 and caspase 3 were quantified by immunohistochemistry after six days. There was no difference in axonal outgrowth between healthy and diabetic rats. However, the sciatic nerve in the diabetic rats exhibited a larger number of ATF 3 expressing Schwann cells at the site of the lesion and also a higher number of caspase 3 expressing Schwann cells. Similar differences were observed in the distal nerve segment between the healthy and diabetic rats. There were no correlations between the number of Schwann cells expressing ATF 3 and caspase 3. Thus, diabetic BB rats display an increased activation of ATF 3 and also a rise in apoptotic caspase 3 expressing Schwann cells, but with no discrepancy in length of axonal outgrowth after nerve injury and repair at six days. Knowledge about signal transduction mechanisms in diabetes after stress may provide new insights into the development of diabetic neuropathy and neuropathic pain.
Mesenchymal stem cells (MSCs) represent a promising therapeutic approach in nerve tissue engineering. To date, the local implantation of MSC in injured nerves has been the only route of administration used. In case of multiple sites of injury, the systemic administration of cells capable of reaching damaged nerves would be advisable. In this regard, we found that an intravenous administration of adipose-derived MSC (ASC) 1 week after sciatic nerve crush injury, a murine model of acute axonal damage, significantly accelerated the functional recovery. Sciatic nerves from ASC-treated mice showed the presence of a restricted number of undifferentiated ASC together with a significant improvement in fiber sprouting and the reduction of inflammatory infiltrates for up to 3 weeks. Besides the immune modulatory effect, our results show that ASC may contribute to peripheral nerve regeneration because of their ability to produce in culture neuroprotective factors such as insulin-like growth factor I, brain-derived neurotrophic factor, or basic fibroblast growth factor. In addition to this production in vitro, we interestingly found that the concentration of glial-derived neurotrophic factor (GDNF) was significantly increased in the sciatic nerves in mice treated with ASC. Since no detectable levels of GDNF were observed in ASC cultures, we hypothesize that ASC induced the local production of GDNF by Schwann cells. In conclusion, we show that systemically injected ASC have a clear therapeutic potential in an acute model of axonal damage. Among the possible mechanisms promoting nerve regeneration, our results rule out a process of trans-differentiation and rather suggest the relevance of a bystander effect, including the production of in situ molecules, which, directly or indirectly through a cross-talk with local glial cells, may modulate the local environment with the down-regulation of inflammation and the promotion of axonal regeneration.
The goal of this study was to quantify the histological changes in the dorsal root ganglion (DRG) and the sciatic nerve in rats subjected to sciatic nerve crush (SNC) following curcumin treatment. The rats were divided into four groups, each including five animals, and underwent the following intervention: group I: control animals which received olive oil; group II: sham-operated animals whose skin of the posterior thigh was opened, sutured, and received the vehicle; group III: SNC animals which received the vehicle; and group IV: SNC plus curcumin (100 mg/kg/day) solved in the vehicle. On the 28th day, the fifth lumbar DRG and sciatic nerve were removed. Volume of the ganglion, mean cell volume, total volume of DRG cells (A- and B-cells), and total surface of DRG cells, total number, diameter, and area of the myelinated nerve fibers were estimated using stereological methods. Except for the volume of the ganglion, all other parameters were decreased after nerve crush. In curcumin-treated rats, these parameters decreased, but to a lesser extent, and the values were significantly higher than in the non-treated SNC group (p<0.04). It can be concluded that in rats after crush, curcumin has a protective effect on the DRG and sciatic nerve.
We have recently described the sequence of functional and morphologic changes occurring after a standardized sciatic nerve crush injury. An 8-week post-injury time was used because this end point is the far most used. Unexpectedly, both functional and morphological data revealed that animals had still not recovered to normal pre-injury levels. Therefore, the present study was designed in order to prolong the observation up to 12 weeks. Functional recovery was evaluated using sciatic functional index (SFI), static sciatic index (SSI), extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. In addition, quantitative morphology was carried out on regenerated nerve fibers. A full functional recovery was predicted by SFI/SSI, EPT and WRL but not all ankle kinematics parameters. Moreover, only two morphological parameters (myelin thickness/axon diameter ratio and fiber/axon diameter ratio) returned to normal values. Data presented in this paper provide a baseline for selecting the adequate end-point and methods of recovery assessment for a rat sciatic nerve crush study and suggest that the combined use of functional and morphological analysis should be recommended in this experimental model.
The effect of hyperbaric oxygen (HBO) treatment on regeneration of the rat sciatic nerve was studied. The sciatic nerve was crushed with a pair of pliers and the animals were either left untreated or subjected to a series of 45-min exposures to 100% O2 at 3.3 atm absolute pressure at 0, 4, and 8 h postoperatively and then every 8 h. Regeneration was evaluated using the pinch-reflex test at 3, 4, or 5 days following surgery and with neurofilament staining at 4 days. The regeneration distances at all time points were significantly longer in animals exposed to hyperbaric oxygen treatment independent of the evaluation procedure. A short initial period of the same HBO treatment schedule, with no more treatments after 25 h, appeared as effective as when treatments were maintained being given every 8 h until evaluation. We conclude that HBO treatment stimulates axonal outgrowth following a nerve crush lesion.
Erythropoietin is a naturally occurring hormone with multiple effects on a number of different cell types. Recent data have suggested neuroprotective and perhaps even neurotrophic roles for erythropoietin. We hypothesized that these functional effects could be demonstrable in standard models of peripheral nerve injury.
Experiments were undertaken to evaluate the effect of erythropoietin on the previously reported standard course of healing of sciatic injuries in mice. The injury groups included mice that were subjected to (1) sham surgery, (2) a calibrated sciatic crush injury, (3) transection of the sciatic nerve followed by epineural repair, or (4) a transection followed by burial of the proximal stump in the adjacent muscle tissue (neurectomy). Either erythropoietin or saline solution was administered to the mice in each of these experimental groups twenty-four hours preinjury, immediately after surgical creation of the injury, twenty-four hours postinjury, or one week postinjury. All mice were evaluated on the basis of the published model for recovery of sciatic nerve motor function by measuring footprint parameters at specific times after the injury. Immunohistochemistry was also performed to assess the erythropoietin-receptor expression profile at the site of injury.
In general, the mice treated with erythropoietin recovered sciatic nerve motor function significantly faster than did the untreated controls. This conclusion was based on a sciatic function index that was 60% better in the erythropoietin-treated mice at seven days postinjury (p < 0.05). Although the group that had been given the erythropoietin immediately postinjury showed the best enhancement of recovery, the timing of the administration of the drug was not critical. Histological analysis demonstrated enhanced erythropoietin-receptor positivity in the nerves that recovered fastest, suggesting that accelerated healing correlates with expression of the receptor in nerve tissue.
Erythropoietin treatment of an acute sciatic nerve crush injury leads to an effect consistent with functional neuroprotection. This protective effect may have clinical relevance, especially since it was detectable even when erythropoietin had been administered up to one week after injury.
Quantification of peripheral nerve regeneration in animal studies of nerve injury and repair by histologic, morphologic, and electrophysiologic parameters has been controversial because such studies may not necessarily correlate with actual nerve function. This study modifies the previously described sciatic functional index (SFI), tibial functional index (TFI), and peroneal functional index (PFI) based on multiple linear regression analysis of factors derived from measurements of walking tracks in rats with defined nerve injuries. The factors that contributed to these formulas were print-length factor (PLF), toe-spread factor (TSF), and intermediary toe-spread factor (ITF). It was shown that animals with selective nerve injuries gave walking tracks that were consistent, predictable, and based on known neuromuscular deficits. The new formula for sciatic functional index was compared with previously described indices. The sciatic functional index, tibial functional index, and peroneal functional index offer the peripheral nerve investigator a noninvasive quantitative assessment of hindlimb motor function in the rat with selective hindlimb nerve injury.
All forms of oxygen deprivation act as a stimulus for the production of the hormone erythropoietin. The main sites of production are specialized renal fibroblasts in adult mammals and hepatic cells in mammalian fetuses and neonates. The hormone's name is a succint description of its main effect: erythropoietin stimulates red cell production from bone marrow precursors and hence controls the O2-carrying capacity of the blood. The peripheral red cell count is kept constant by a closely controlled feedback mechanism involving O2 supply, erythropoietin secretion and erythropoiesis; the system may become unbalanced in conditions such as chronic renal disease, chronic inflammation and prematurity. Recombinant human erythropoietin is used as hormonal replacement therapy to correct various types of anemia and replenish the red cell count following hemorrhage or blood donation for autologous transfusion.
In vitro granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin (EPO), and erythroid differentiation factor (EDF) augmented choline acetyltransferase (ChAT) activity in mouse embryonic primary septal neurons and in cholinergic hybridoma cell line, SN6.10.2.2. This is similar to the effects seen with interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Moreover, in vivo GM-CSF and EPO promoted survival of septal cholinergic neurons in adult rats which had undergone fimbria-fornix transections. These results suggest that some of the hematopoietic factors act on cholinergic neurons as 'neurotrophic factors' to influence the differentiation, maintenance and regeneration of these neurons.
Recently, erythropoietin has been shown to be produced by astrocytes and its production is hypoxia-inducible. In the present study, we demonstrated, using a reverse transcription-polymerase chain reaction assay and immunostaining of the cells, that the erythropoietin receptor was expressed in cultured hippocampal and cerebral cortical neurons of day 19 rat embryo. Erythropoietin protected the cultured neurons from glutamate neurotoxicity. Neurons cultured for seven to 10 days were exposed to glutamate for 15 min and after culture for a further 24 h in the absence of glutamate the neuron survival was assayed. Significant protection was observed with erythropoietin from 3 pM (c. 100 pg/ml) in a dose-dependent manner. The protection was completely reversed by co-application of a soluble erythropoietin receptor, an extracellular domain capable of binding with erythropoietin. For exhibition of the neuroprotective effect, exposure of neurons to erythropoietin approximately 8 h prior to exposure to glutamate was required. Experiments with the inhibitors indicated that RNA and protein syntheses were necessary for the protection. However, exposure to erythropoietin for a short period (5 min or less) was sufficient to elicit the protective effect. The protective effect of erythropoietin was blocked by the simultaneous addition of EGTA. These findings and the previous finding that erythropoietin induces a rapid and transient increase in intracellular Ca2+ concentration in neuronal cells suggest that erythropoietin plays a neuroprotective role in brain injury caused by hypoxia or ischemia and that erythropoietin-induced Ca2+ influx from outside of the cells is a critical initial event yielding an enhanced resistance of the neurons to glutamate toxicity.
Exogenic nerve growth factor in the crushed nerve of rat promotes the development of reparative-regenerative processes. This was established by electron microscopy and electrophysiological method and is expressed through the activation of phagocytosis in the remained crushed axons in distal segments, relatively more early and vast penetration of the growing fibres to the outside of the traumatic zone, a single supramaximum stimulation of proximal region above the appearance and growth of action potential amplitude and also its spreading to the periphery as compared to control animals, treated with equal amounts of isotonic natrium chloride.
Erythropoietin (Epo), which is produced by the kidney in the adult and by the liver in the fetus, increases red blood cells by supporting the survival of erythroid progenitor cells and stimulating their differentiation and proliferation via binding to Epo receptor (EpoR). The main signal in the control of Epo production is oxygen; hypoxia stimulates Epo production through activation of Epo gene transcription. Tremendous progress in our understanding of molecular mechanisms of Epo action on erythroid cells and regulation of the Epo production has been made by manipulation of cDNAs and genes of Epo and EpoR. Studies on hypoxic induction of Epo gene transcription led to the identification of hypoxia-inducible factor (HIF-1), a transcriptional factor, that functions as a global regulator of hypoxic gene expression. Paracrine Epo/EpoR systems that are independent of the endocrine erythropoietic system (kidney/bone marrow) have been found in the central nervous system and uterus. Novel functions of Epo at these local sites and tissue-specific regulation of Epo production including a newly found potent regulator (estrogen) have been proposed. The tissue-specific regulation rationalizes the specific functions of Epo produced by individual tissues.
Eleven thousand Americans each year are affected by paralysis, a devastating injury that possesses associated annual costs of $7 billion (American Paralysis Association, 1997). Currently, there is no effective treatment for damage to the central nervous system (CNS), and acute spinal cord injury has been extraordinarily resistant to treatment. Compared to spinal cord injury, damage to peripheral nerves is considerably more common. In 1995, there were in excess of 50,000 peripheral nerve repair procedures performed. (National Center for Health Statistics based on Classification of Diseases, 9th Revision, Clinical Modification for the following categories: ICD-9 CM Code: 04.3, 04.5, 04.6, 04.7). These data, however, probably underestimate the number of nerve injuries appreciated, as not all surgical or traumatic lesions can be repaired. Further, intraabodominal procedures may add to the number of neurologic injuries by damage to the autonomic system through tumor resection. For example, studies assessing the outcome of impotency following radical prostatectomy demonstrated 212 of 503 previously potent men (42%) suffered impotency when partial or complete resection of one or both cavernosal nerve(s). This impotency rate decreased to 24% when the nerves were left intact (Quinlan et al., J. Urol. 1991;145:380-383; J. Urol. 1991;145:998-1002).
Standardized experimental nerve crush attempts should include the number, duration, and intensity (amount of pressure) of crushes. The authors have developed a new crushing device, a clamp with which predetermined forces can be applied to nerves. This allows the exertion of different, standardized forces to crush a nerve within a scale that produces second-degree injuries. The main advantages of the clamp are that it is small, although very robust, is purely mechanical, and is easy to handle. The jaws of the clamp are not serrated, so that pressure on the nerve is uniformly transmitted. To avoid unintended nerve damage, the edges of the jaws are smoothly rounded off. The closure of the clamp is mechanized by a spring. As the spring is exchangeable, any number of different preloads are available. The force can be varied, according to different requirements, and is applicable to variantly thick nerves in any experimental animal, thus enhancing standardization, and making cross-over comparisons of experimental study results possible.
Erythropoietin (Epo) has been shown to have potent anti-apoptotic activity in central nervous system neurons in animal models of ischaemic injury. Recently, Epo and its receptor (EpoR) have been identified in the peripheral nervous system [Campana & Myers (2001), FASEB J., 15, 1804-1806]. Herein, we demonstrate that in painful neuropathy caused by L5 spinal nerve crush (SNC), therapy with recombinant human Epo (rhEpo) reduced dorsal root ganglion (DRG) apoptosis and pain behaviours. Quantification of both DRG neurons and satellite cells revealed that vehicle-treated, crush-injured DRGs had 35.5 +/- 8.3% apoptotic neurons and 23.5 +/- 2.36% satellite cells compared with 7.5 +/- 6.3% apoptotic neurons and 6.4 +/- 3.94% satellite cells in rhEpo-treated, crush-injured DRGs (P < 0.05). While rhEpo-treated animals were not initially protected from mechanical allodynia associated with L5 SNC, rhEpo did significantly improve recovery rates compared to vehicle-treated animals (P < 0.01). Systemic rhEpo therapy increased JAK2 phosphorylation, a key anti-apoptotic signalling molecule for Epo-induced neuroprotection, in DRGs after crush. Dual immunofluorescence demonstrated Epo-induced JAK2-p was associated with both neuronal and glial cells. JAK2-p was associated with NF200-positive large neurons and with smaller neurons. This population of small neurons did not colocalize with IB4, a marker of nonpeptidergic, glial derived growth factor-responsive neurons. The findings link anti-apoptosis activities of Epo/EpoR/JAK2 in DRG neurons capable of inducing protracted pain states with reductions in pain behaviours, and therefore support a role for Epo therapy in the treatment of neuropathic pain.
The authors investigated the association of L5 proximal nerve root injury with spinal cord neuronal apoptosis (histologic) and whether exogenous erythropoietin therapy might reduce apoptosis/or pain (behavioral).
The first objective was to determine whether nerve root crush induces specific programmed cell death of spinal neurons in the dorsal and ventral horn and whether this is correlated with pain behaviors. The second objective was to determine if exogenous erythropoietin might reduce apoptosis and/or pain.
Whether spinal neuronal apoptosis is correlated with pain behaviors following nerve root injury remains unknown. It has been hypothesized that neuroprotective factors may alleviate pain behaviors by protecting neurons from death. Erythropoietin is a hematopoietic growth factor that recently has been demonstrated as a potent neuroprotective factor against ischemic damage in the brain. The effects of erythropoietin on pain and spinal cord neurons have not been examined.
Sprague-Dawley rats received a L5 proximal nerve root crush injury or sham operation and were separated into two treatment groups for subcutaneous injection: 1) vehicle; 2) recombinant human erythropoietin, 2680 U/kg. The rats were sacrificed, and spinal cords were removed for apoptotic and immunohistochemical analysis at 0, 1, and 3 days after surgery. To determine whether recombinant human erythropoietin prevented mechanical allodynia in animals with nerve root crushes (n = 12/group), both treatment groups were tested for pain behaviors using the von Frey test at -1, -2, -3, 1, 3, 7, 11, and 14 days after surgery.
After nerve root injury, apoptotic neurons increased by 80% in the ipsilateral spinal cord and moderately in contralateral spinal cord in vehicle-treated animals compared to uninjured controls. Recombinant human erythropoietin reduced (P < 0.05) neuronal apoptosis in both superficial dorsal and ventral horns of the spinal cord. This corresponded with identification of erythropoietin and its receptors on spinal neurons and reductions in TNF-alpha colocalization in ventral horn neurons. Mechanical allodynia developed in the corresponding ipsilateral hind paw within 1 day and was sustained until day 14. Recombinant human erythropoietin-treated animals demonstrated faster recovery from mechanical allodynia compared with vehicle-treated controls (P < 0.05).
Our findings indicated that L5 proximal nerve root crush increased neuronal apoptosis in the superficial dorsal and ventral horn that correlated with mechanical allodynia. Exogenous recombinant human erythropoietin facilitated receptor-mediated neuroprotection of spinal cord neurons and faster recovery from mechanical allodynia. Erythropoietin may be a potential therapeutic factor for patients with low back pain by providing pain relief and neuroprotection.
Life science investigators must be prepared to continuously change their minds about biological processes and functions during their career, in order to keep pace with evolving concepts and breakthrough discoveries. Apoptosis is one of the areas of biology in which this mental flexibility is most needed, as there has been considerable progress over the past few years. The review presented in this issue of CDD by Ghezzi and Brines1 provides an example of this, by showing how an old cytokine – previously known, and commercially exploited, for its unique role in the proliferation and differentiation of the haematopoietic system – has revealed an unsuspected function as an antiapoptotic factor throughout the body, particularly in the survival of neurons.
Patients with injured hands and forearms of varying severity [Hand Injury Severity Score (HISS)] were studied prospectively, including analysis of costs, hand/arm function (DASH), and health status (SF-36). Costs, duration of sick-leave, DASH-score (high score; impaired function) increased by severity of injury (higher HISS) and the greatest proportion of total costs resulted from lost production. Most employed patients returned to work within a year, but even minor injuries were expensive. HISS and costs of care during an emergency were significantly associated with duration of sick-leave, although HISS did not fully explain variation in costs and duration of sick-leave. DASH-score at one year was associated with variation in age, HISS, and residual health care costs. Results of DASH and subgroups for physical and bodily pain on SF-36 were consistent. Injuries to hand and forearm may generate high costs for society in terms of health care and long periods of sick-leave (lost production), but even minor injuries should be accounted for.
The rat model of cavernous nerve (CN) injury has been developed in an effort to define the functional and structural consequences of neural trauma in the corpus cavernosum. However, there is no universally accepted method of inducing nerve injury in this model, with neurotomy and crush models being used currently. To address this issue, we induced CN injury using various techniques in an effort to compare the hemodynamic sequelae of these injuries.
Twenty-five adult male Sprague-Dawley rats were divided into five groups: (1) control: laparotomy only; (2) exposure: laparotomy and exposure of cavernous nerves bilaterally without nerve manipulation; (3) neurotomy; bilateral neurotomy; (4) bulldog crush: bilateral nerve crush with bulldog vascular clamp; and (5) hemostat nerve crush: bilateral nerve crush with a hemostat. Ten days later, a second operation was performed during which systemic mean arterial pressure (MAP) and intracavernosal pressure (ICP) were measured in response to CN stimulation proximal to the site of injury. Hemodynamic endpoints assessed included ICP/MAP ratio, rate of tumescence, and rate of detumescence.
The ICP/MAP ratio (mean +/- 95% confidence interval) in the control group was 70 +/- 4%. ICP/MAP ratios were significantly reduced in all CN injury groups compared with control group: exposure: 41 +/- 10% (P < 0.001); neurotomy: 35 +/- 15% (P < 0.001); bulldog crush: 39 +/- 13% (P < 0.001); hemostat crush: 31 +/- 9% (P < 0.0001). No significant difference existed in ICP/MAP ratios between the injury groups. Of note, the exposure group also demonstrated significant functional alterations. The rates of tumescence and detumescence were significantly reduced in all groups compared with the control group.
No significant difference in the magnitude and consistency of hemodynamic alterations has been demonstrated in all CN injury models assessed in this study.