Activity cage as a method to analyze functional recovery after sciatic nerve injury in mice

The aim of this paper is to show the activity cage as a viable method for tracking functional nerve recovery. The activity cage measures spontaneous coordinate activity, meaning movement in either the horizontal or vertical plane, of experimental animals within a specified amount of time. This uses a minimum of researcher time conducting functional testing to determine functional recovery of the nerve. Using microsurgical forceps, a crush injury was inflicted unilaterally, on the left side, upon the 4-month-old C3H mice creating a very high degree of pressure for 6 s upon the exposed sciatic nerve. The locomotion function of the mice was evaluated using the activity cage preoperatively, 1, 7, 14, 21, and 28 days after the surgical procedure. We found that using the activity cage functional recovery occurred by 14 days after nerve crush injury. It was also shown that, coinciding with functional recovery, immunohistochemistry changes for GD1a and nNOS appeared at the level of L4, where the sciatic nerve joins the spinal column. GD1a and nNOS have both been linked to regenerative processes in mammalian nervous systems.     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

Acetyl salicylic acid augments functional recovery following sciatic nerve crush in mice

The Department of Physical Medicine, Rehabilitation and Electrodiagnosis of Shaheed Beheshti Medical University in collaboration with the Iranian Society of Physical Medicine and Rehabilitation (ISPMR) held the 1^st Congress of Electrodiagnostic Medicine in Peripheral Nerve Lesions on December 21–22, 2006. Electrodiagnostic medicine is a specific branch of medicine used by specialist physicians in the field of physical medicine and rehabilitation and/or neurology to diagnose, prognosticate and plan treatment options of peripheral nerve lesions. This meeting was hold to discuss multidisciplinary approaches to this common and important topic in the medical field.     

Faster nerve regeneration after sciatic nerve injury in mice over-expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF-2) is expressed in the peripheral nervous system and is up-regulated after nerve lesion. It has been demonstrated that administration of FGF-2 protects neurons from injury-induced cell death and promotes axonal regrowth. Using transgenic mice over-expressing FGF-2 (TgFGF-2), we addressed the importance of endogenously generated FGF-2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild-type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF-2. Morphometric evaluation of intact nerves from TgFGF-2 mice revealed no difference in number and size of myelinated fibers compared to wild-type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF-2 over-expression on Schwann cell proliferation during the early regeneration process, we used BrdU-labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild-types. We propose that endogenously synthesized FGF-2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination.     

Faster nerve regeneration after sciatic nerve injury in mice over‐expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF‐2) is expressed in the peripheral nervous system and is up‐regulated after nerve lesion. It has been demonstrated that administration of FGF‐2 protects neurons from injury‐induced cell death and promotes axonal regrowth. Using transgenic mice over‐expressing FGF‐2 (TgFGF‐2), we addressed the importance of endogenously generated FGF‐2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild‐type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF‐2. Morphometric evaluation of intact nerves from TgFGF‐2 mice revealed no difference in number and size of myelinated fibers compared to wild‐type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF‐2 over‐expression on Schwann cell proliferation during the early regeneration process, we used BrdU‐labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild‐types. We propose that endogenously synthesized FGF‐2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Side distinct sciatic nerve recovery differences between rats and mice

The Sciatic Functional Index (SFI) is widely used to evaluate functional recovery after sciatic nerve injury, primarily in the rat, and more recently shown useful in the mouse. This quantitative, non-invasive method allows tracking of regeneration capability, visible in the gait of the animal. Using a Martin micro needle holder, carrying a force measured to be 49.2 N, the left sciatic nerve was crushed for 60 s. We accumulated data from walking tracks collected preoperatively and 1, 7, 14, 21, and 28 days after injury. SFI values were first calculated in the traditional manner. Then using the preoperative values as the normal value in the postoperative calculations, SFI was again calculated; this isolated the calculations to either injured or contra lateral leg giving a “split” plot. The traditional SFI calculations resulted in typical shaped graphs for both rats and mice. However, the “split” SFI calculations showed how rats and mice differ in their recovery from sciatic nerve injury. The mouse graph shows the intact leg remaining stable and the injured leg having functional impairment, which then recovers. The rat graph showed functional impairment of the injured leg, however, the intact leg had an increase in SFI values as if to compensate until the injured leg showed recovery.     

Side distinct sciatic nerve recovery differences between rats and mice

The Sciatic Functional Index (SFI) is widely used to evaluate functional recovery after sciatic nerve injury, primarily in the rat, and more recently shown useful in the mouse. This quantitative, non-invasive method allows tracking of regeneration capability, visible in the gait of the animal. Using a Martin micro needle holder, carrying a force measured to be 49.2 N, the left sciatic nerve was crushed for 60 s. We accumulated data from walking tracks collected preoperatively and 1, 7, 14, 21, and 28 days after injury. SFI values were first calculated in the traditional manner. Then using the preoperative values as the normal value in the postoperative calculations, SFI was again calculated; this isolated the calculations to either injured or contra lateral leg giving a "split" plot. The traditional SFI calculations resulted in typical shaped graphs for both rats and mice. However, the "split" SFI calculations showed how rats and mice differ in their recovery from sciatic nerve injury. The mouse graph shows the intact leg remaining stable and the injured leg having functional impairment, which then recovers. The rat graph showed functional impairment of the injured leg, however, the intact leg had an increase in SFI values as if to compensate until the injured leg showed recovery.     

Lentiviral vectors enveloped with rabies virus glycoprotein can be used as a novel retrograde tracer to assess nerve recovery in rat sciatic nerve injury models

Retrograde labeling has become the new “gold standard” technique to evaluate the recovery of injured peripheral nerves. In this study, lentiviral vectors with rabies virus glycoprotein envelop (RABV-G-LV) and RFP genes are injected into gastrocnemius muscle to determine the location of RFP in sciatic nerves. We then examine RFP expression in the L4-S1 spinal cord and sensory dorsal root ganglia and in the rat sciatic nerve, isolated Schwann cells, viral dose to expression relationship and the use of RABV-G-LV as a retrograde tracer for regeneration in the injured rat sciatic nerve. VSV-G-LV was used as control for viral envelope specificity. Results showed that RFP were positive in the myelin sheath and lumbar spinal motorneurons of the RABV-G-LV group. RFP gene could be detected both in myelinated Schwann cells and lumbar spinal motor neurons in the RABV-G-LV group. Schwann cells isolated from the RABV-G-LV injected postnatal Sprague Dawley rats were also RFP-gene positive. All the results obtained in the VSV-G-LV group were negative. Distribution of RFP was unaltered and the level of RFP expression increasing with time progressing. RABV-G-LV could assess the amount of functional regenerating nerve fibers two months post-operation in the four models. This method offers an easy-operated and consistent standardized approach for retrograde labeling regenerating peripheral nerves, which may be a significant supplement for the previous RABV-G-LV-related retrograde labeling study.     

Excitability changes in the sciatic nerve and triceps surae muscle after spinal cord injury in mice

Background

Ischemia reperfusion (I/R) is common in various pathological conditions like diabetic complication, rheumatic arthritis, necrotizing vascular occlusive disease and trauma.

Methods

We have evaluated the effect of tacrolimus (1, 2 and 3 mg/kg, p.o. for 10 consecutive days) on femoral arterial ischemic reperfusion (I/R) induced neuropathic pain in rats. Behavioral parameters (i.e. hot plate, radiant heat, acetone drop, tail heat hyperalgesia, tail flick and tail cold allodynia tests) were assessed at different time intervals (i.e. 0, 1, 4, 7, 10, 13 and 16^th day) and biochemical analysis in serum and tissue samples were also performed along with histopathological studies.

Results

Behavioral pain assessment revealed increase in the paw and tail withdrawal threshold in tacrolimus treated groups against hyperalgesic and allodynic stimuli as compared to the sham control group. We observed a decrease in the serum nitrate and thiobarbituric acid reactive substance (TBARS) levels along with reduction in tissue myeloperoxidase (MPO) and total calcium levels, whereas, rise in tissue reduced glutathione levels in tacrolimus treated groups. However, significant results were obtained in medium and high dose treated group as compared to sham control group. Histopathological study had revealed the increase in the neuronal edema and axonal degeneration in the I/R group whereas, tacrolimus ameliorate these effects.

Conclusion

Our results indicate the anti-oxidative, anti-inflammatory and calcium modulatory actions of tacrolimus. Therefore, it can be used as a therapeutic agent for the treatment of vascular inflammatory related neuropathic pain.     

Increased expression of Gem after rat sciatic nerve injury

Gem belongs to the Rad/Gem/Kir subfamily of Ras-related GTPases, whose expression is induced in several cell types upon activation by extracellular stimuli. Two functions of Gem have been demonstrated, including regulation of voltage-gated calcium channel activity and inhibition of Rho kinase-mediated cytoskeletal reorganization, such as stress fiber formation and neurite retraction. Because of the essential relationship between actin reorganization and peripheral nerve regeneration, we investigated the spatiotemporal expression of Gem in a rat sciatic nerve crush (SNC) model. After never injury, we observed that Gem had a significant up-regulation from 1 day, peaked at day 5 and then gradually decreased to the normal level. At its peak expression, Gem expressed mainly in Schwann cells (SCs) and macrophages of the distal sciatic nerve segment, but had few colocalization in axons. In addition, the peak expression of Gem was in parallel with PCNA, and numerous SCs expressing Gem were PCNA positive. Thus, all of our findings suggested that Gem may be involved in the pathophysiology of sciatic nerve after SNC.     

GDNF-ADSCs-APG embedding enhances sciatic nerve regeneration after electrical injury in a rat model

The pluripotency of adipose-derived stem cells (ADSCs) makes them appropriate for tissue repair and wound healing. Owing to the repair properties of autologous platelet–rich gel (APG), which is based on easily accessible blood platelets, its clinical use has been increasingly recognized by physicians. The aim of this study was to investigate the effect of combined treatment with ADSCs and APG on sciatic nerve regeneration after electrical injury. To facilitate the differentiation of ADSCs, glial cell line–derived neurotrophic factor (GDNF) was overexpressed in ADSCs by lentivirus transfection. GDNF-ADSCs were mingled with APG gradient concentrations, and in vitro, cell proliferation and differentiation were examined with 5-ethynyl-2′-deoxyuridine staining and immunofluorescence. A rat model was established by exposing the sciatic nerve to an electrical current of 220 V for 3 seconds. Rat hind-limb motor function and sciatic nerve regeneration were subsequently evaluated. Rat ADSCs were characterized by high expression of CD90 and CD105, with scant expression of CD34 and CD45. We found that GDNF protein expression in ADSCs was elevated after Lenti-GDNF transfection. In GDNF-ADSCs-APG cultures, GDNF was increasingly produced while tissue growth factor-β was reduced as incubation time was increased. ADSC proliferation was augmented and neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) expression were upregulated in GDNF-ADSCs-APG. In addition, limb motor function and nerve axon growth were improved after GDNF-ADSCs-APG treatment. In conclusion, our study demonstrates the combined effect of ADSCs and APG in peripheral nerve regeneration and may lead to treatments that benefit patients with electrical injuries.     

Photobiomodulation at a wavelength of 633 nm leads to faster functional recovery than 804 nm after facial nerve injury

We analyzed the effects of photobiomodulation (PBM) of various wavelengths on regeneration of the facial nerve using in vitro and in vivo experimental models. We assessed the antioxidative effect of PBM in geniculate ganglion neurons irradiated with a diode laser at 633 nm, 780 nm and 804 nm. Wavelengths of 633 and 780 nm but not 804 nm inhibited cell death by oxidative stress. We assessed the effects of PBM on functional and morphologic recovery in rats divided into control, facial nerve damage (FND) and FND irradiated with a 633 nm or 804 nm lasers. Injured rats treated with 633-nm light had better facial palsy scores, larger axon diameter and higher expression of Schwann cells compared with the FND group. No positive results were observed in rats irradiated at 804-nm light. These findings indicate that 633-nm PBM promotes accelerated nerve regeneration and improved functional recovery in an injured facial nerve.     

Neurotrophic factors and their receptors in axonal regeneration and functional recovery after peripheral nerve injury

Over a half a century of research has confirmed that neurotrophic factors promote the survival and process outgrowth of isolated neurons in vitro. The mechanisms by which neurotrophic factors mediate these survival-promoting effects have also been well characterized. In vivo, peripheral neurons are critically dependent on limited amounts of neurotrophic factors during development. After peripheral nerve injury, the adult mammalian peripheral nervous system responds by making neurotrophic factors once again available, either by autocrine or paracrine sources. Three families of neurotrophic factors were compared, the neurotrophins, the GDNF family of neurotrophic factors, and the neuropoetic cytokines. Following a general overview of the mechanisms by which these neurotrophic factors mediate their effects, we reviewed the temporal pattern of expression of the neurotrophic factors and their receptors by axotomized motoneurons as well as in the distal nerve stump after peripheral nerve injury. We discussed recent experiments from our lab and others which have examined the role of neurotrophic factors in peripheral nerve injury. Although our understanding of the mechanisms by which neurotrophic factors mediate their effects in vivo are poorly understood, evidence is beginning to emerge that similar phenomena observed in vitro also apply to nerve regeneration in vivo.     

Changes in CLIP3 expression after sciatic nerve injury in adult rats

CLIP3 (cytoplasmic linker protein 3) is a 547 amino acid residue cytoplasmic protein that localises to Golgi stacks and tubulovesicular elements juxtaposed to Golgi cisternae. Composed of three Ank (ankyrin) repeats and two CAP-Gly (cytoskeleton-associated protein-glycine) domains, CLIP3 may function as a cytoplasmic linker protein that is involved in TGN–endosome dynamics. To define the expression and role of CLIP3 during peripheral nervous system degeneration and regeneration, we created an acute sciatic nerve injury (SNI) model in adult rats. Western blot analyses revealed prominent up-regulation of CLIP3 and PCNA (proliferating cell nuclear antigen) protein levels at 3?days after SNI. Immunohistochemistry displayed that the expression of CLIP3 was noticeably increased in the injured nerve. Immunofluorescence further revealed that the CLIP3 and PCNA proteins colocalised respectively with S100 in the cytoplasm of Schwann cells. The expression profile of the SC/neuron co-cultures demonstrated that CLIP3 and PCNA protein levels were markedly expressed during the early stage of myelination. These results suggest that CLIP3 is likely associated with the myelination of proliferating Schwann cells, and nerve tissue regeneration after peripheral nerve injury. CLIP3 and PCNA expression during early myelination may be related to the direct uptake and transport of lipids and cholesterol, which were derived from the degenerating myelin, by Schwann cells to prepare for the formation of myelin sheath-like structures around regenerated axons after SNI.     

Intraperitoneal Alpha-Lipoic Acid to prevent neural damage after crush injury to the rat sciatic nerve

The hypothesis is explored that CRPS I (the "new" RSD) persists due to undiagnosed injured joint afferents, and/or cutaneous neuromas, and/or nerve compressions, and is, therefore, a misdiagnosed form of CRPS II (the "new" causalgia). An IRB-approved, retrospective chart review on a series of 100 consecutive patients with "RSD" identified 40 upper and 30 lower extremity patients for surgery based upon their history, physical examination, neurosensory testing, and nerve blocks. Based upon decreased pain medication usage and recovery of function, outcome in the upper extremity, at a mean of 27.9 months follow-up (range of 9 to 81 months), gave results that were excellent in 40% (16 of 40 patients), good in 40% (16 of 40 patients) and failure 20% (8 of 40 patients). In the lower extremity, at a mean of 23.0 months follow-up (range of 9 to 69 months) the results were excellent in 47% (14 of 30 patients), good in 33% (10 of 30 patients) and failure 20% (6 of 30 patients). It is concluded that most patients referred with a diagnosis of CRPS I have continuing pain input from injured joint or cutaneous afferents, and/or nerve compressions, and, therefore, similar to a patient with CRPS II, they can be treated successfully with an appropriate peripheral nerve surgical strategy.     

Absence of mechanical allodynia and Abeta-fiber sprouting after sciatic nerve injury in mice lacking membrane-type 5 matrix metalloproteinase

Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade extracellular matrix components. Membrane-type 5 MMP (MT5-MMP/MMP-24) was identified as neuron-specific, and is believed to contribute to neuronal circuit formation and plasticity. To elucidate its function in vivo, we have generated mice lacking MT5-MMP by gene targeting. MT5-MMP-deficient mice were born without obvious morphological abnormalities. No apparent histological defects were observed in the nervous system either. However, MT5-MMP-deficient mice did not develop neuropathic pain with mechanical allodynia after sciatic nerve injury, though responses to acute noxious stimuli were normal. Neuropathic pain induced by peripheral nerve lesions is known to accompany structural reorganization of the nervous system. Intraneural injection of cholera toxin B subunit, a transganglionic tracer, into the injured sciatic nerve of wild-type mice revealed that the myelinated Abeta-fiber primary afferents sprouted from laminae III-VI of the dorsal horn of the spinal cord and invaded lamina II. However, no such sprouting and invasion of Abeta-fibers were observed in MT5-MMP-deficient mice. These findings suggest that MT5-MMP is essential for the development of mechanical allodynia and plays an important role in neuronal plasticity in this mouse model.     

Rat sciatic nerve crush injury and recovery tracked by plantar test and immunohistochemistry analysis

An experimental crush injury to the sciatic nerve, with a crush force of 49.2 N (pressure p=1.98x10(8) Pa), was inflicted in 30 male rats (Wistar). A control group (sham), with the same number of rats, was also operated upon exactly as the experimental group but without the crush injury. We tested the sensory and motor recovery of the sciatic nerve with Hargreaves method, using an apparatus from Ugo Basile, Italy. Testing was continued for both legs of each rat, injured and uninjured, starting preoperatively (0 day), and then 1, 7, 14, 21, and 28 days postoperatively. The same experiment was run simultaneously with the sham group. The Plantar test showed recovery of the sensory and motor function of the sciatic nerve, though not complete recovery, by 28 days. An immunohistochemical experiment was run in parallel with the plantar test on L3-L6 segments of the spinal cord from where the sciatic nerve extends. We used antibodies for Myelin-associated glycoprotein (MAG), and gangliosides GD1a and GT1b on the aforesaid part of the spinal cord. The immunohistochemical methods showed changes in sensory and motor axons in the spinal cord segment L3-L6 which suggest correspondence with the results of the Plantar test, in terms of recovery of the sensory and motor function after injury of the sciatic nerve. The immunohistochemical results also show ipsilateral and contralateral changes following injury. Results of the plantar test are suggestive that the rat shows compensation for an injury in its contralateral leg.     

Interleukin-17A regulates ependymal cell proliferation and functional recovery after spinal cord injury in mice

Ependymal cells have been suggested to act as neural stem cells and exert beneficial effects after spinal cord injury (SCI). However, the molecular mechanism underlying ependymal cell regulation after SCI remains unknown. To examine the possible effect of IL-17A on ependymal cell proliferation after SCI, we locally administrated IL-17A neutralizing antibody to the injured spinal cord of a contusion SCI mouse model, and revealed that IL-17A neutralization promoted ependymal cell proliferation, which was paralleled by functional recovery and axonal reorganization of both the corticospinal tract and the raphespinal tract. Further, to test whether ependymal cell-specific manipulation of IL-17A signaling is enough to affect the outcomes of SCI, we generated ependymal cell-specific conditional IL-17RA-knockout mice and analyzed their anatomical and functional response to SCI. As a result, conditional knockout of IL-17RA in ependymal cells enhanced both axonal growth and functional recovery, accompanied by an increase in mRNA expression of neurotrophic factors. Thus, Ependymal cells may enhance the regenerative process partially by secreting neurotrophic factors, and IL-17A stimulation negatively regulates this beneficial effect. Molecular manipulation of ependymal cells might be a viable strategy for improving functional recovery.Subject terms: Neuroimmunology, Spinal cord injury