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.     

Knockout of p75(NTR) impairs re-myelination of injured sciatic nerve in mice

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75(NTR), in activated Schwann cells in the Wallerian degenerated nerve is up-regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75(NTR) in remyelination of the sciatic nerve was investigated in p75(NTR) mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild-type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild-type mice. Real-time RT-PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75(NTR) mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75(NTR) is important for the myelinogenesis during the regeneration of peripheral nerves after injury.     

Progranulin contributes to endogenous mechanisms of pain defense after nerve injury in mice

Progranulin haploinsufficiency is associated with frontotemporal dementia in humans. Deficiency of progranulin led to exaggerated inflammation and premature aging in mice. The role of progranulin in adaptations to nerve injury and neuropathic pain are still unknown. Here we found that progranulin is up-regulated after injury of the sciatic nerve in the mouse ipsilateral dorsal root ganglia and spinal cord, most prominently in the microglia surrounding injured motor neurons. Progranulin knockdown by continuous intrathecal spinal delivery of small interfering RNA after sciatic nerve injury intensified neuropathic pain-like behaviour and delayed the recovery of motor functions. Compared to wild-type mice, progranulin-deficient mice developed more intense nociceptive hypersensitivity after nerve injury. The differences escalated with aging. Knockdown of progranulin reduced the survival of dissociated primary neurons and neurite outgrowth, whereas addition of recombinant progranulin rescued primary dorsal root ganglia neurons from cell death induced by nerve growth factor withdrawal. Thus, up-regulation of progranulin after neuronal injury may reduce neuropathic pain and help motor function recovery, at least in part, by promoting survival of injured neurons and supporting regrowth. A deficiency in this mechanism may increase the risk for injury-associated chronic pain.     

Effects of safranal,a constituent of saffron,and vitamin E on nerve functions and histopathology following crush injury of sciatic nerve in rats

Safranal is one of the major components of saffron and has many biological effects such as antioxidant property. The present study investigated the effects of safranal on sciatic nerve function after induction of crush injury. We also used of vitamin E as a reference potent antioxidant agent.In anesthetized rats, right sciatic nerve was crushed using a small haemostatic forceps. Functional recovery was assessed using sciatic functional index (SFI). Acetone spray and von Frey filament tests were used for neuropathic pain assay. Histopathological changes including severities of Wallerian degeneration of sciatic nerve and gastrocnemius muscle atrophy were investigated by light microscopy. Blood levels of malodialdehyde (MDA) were also measured.The SFI values were accelerated, cold and mechanical allodynia were suppressed, the severities of Wallerian degeneration and muscular atrophy were improved, and the increased MDA level was reversed with 10 consecutive days intraperitoneal injections of 0.2 and 0.8 mg/kg of safranal and 100 mg/kg of vitamin E.It is concluded that safranal and vitamin E produced same improving effects on crushed-injured sciatic nerve functions. Inhibition of oxidative stress pathway may be involved in improving effects of safranal and vitamin E on functions and histopathology of an injured peripheral nerve.     

Conserved Dopamine Neurotrophic Factor-Transduced Mesenchymal Stem Cells Promote Axon Regeneration and Functional Recovery of Injured Sciatic Nerve

Peripheral nerve injury (PNI) is a common disease that often results in axonal degeneration and the loss of neurons, ultimately leading to limited nerve regeneration and severe functional impairment. Currently, there are no effective treatments for PNI. In the present study, we transduced conserved dopamine neurotrophic factor (CDNF) into mesenchymal stem cells (MSCs) in collagen tubes to investigate their regenerative effects on rat peripheral nerves in an in vivo transection model. Scanning electron microscopy of the collagen tubes demonstrated their ability to be resorbed in vivo. We observed notable overexpression of the CDNF protein in the distal sciatic nerve after application of CDNF-MSCs. Quantitative analysis of neurofilament 200 (NF200) and S100 immunohistochemistry showed significant enhancement of axonal and Schwann cell regeneration in the group receiving CDNF-MSCs (CDNF-MSCs group) compared with the control groups. Myelination thickness, axon diameter and the axon-to fiber diameter ratio (G-ratio) were significantly higher in the CDNF-MSCs group at 8 and 12 weeks after nerve transection surgery. After surgery, the sciatic functional index, target muscle weight, wet weight ratio of gastrocnemius muscle and horseradish peroxidase (HRP) tracing demonstrated functional recovery. Light and electron microscopy confirmed successful regeneration of the sciatic nerve. The greater numbers of HRP-labeled neuron cell bodies and increased sciatic nerve index values (SFI) in the CDNF-MSCs group suggest that CDNF exerts neuroprotective effects in vivo. We also observed higher target muscle weights and a significant improvement in muscle atrophism in the CDNF-MSCs group. Collectively, these findings indicate that CDNF gene therapy delivered by MSCs is capable of promoting nerve regeneration and functional recovery, likely because of the significant neuroprotective and neurotrophic effects of CDNF and the superior environment offered by MSCs and collagen tubes.     

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.     

Use of poly(DL-lactide-ε-caprolactone) membranes and mesenchymal stem cells from the Wharton's jelly of the umbilical cord for promoting nerve regeneration in axonotmesis: In vitro and in vivo analysis

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).     

Potentiation of angiogenesis and regeneration by G-CSF after sciatic nerve crush injury

Granulocyte colony-stimulating factor (G-CSF) demonstrates neuroprotective effects through different mechanisms, including mobilization of bone marrow cells. However, the influence of G-CSF-mediated mobilization of bone marrow-derived cells on injured sciatic nerves remains to be elucidated. The administration of G-CSF promoted a short-term functional recovery 7 days after crush injury in sciatic nerves. A double-immunofluorescence study using green fluorescent protein-chimeric mice revealed that bone marrow-derived CD34+ cells were predominantly mobilized and migrated into injured nerves after G-CSF treatment. G-CSF-mediated beneficial effects against sciatic nerve injury were associated with increased CD34+ cell deposition, vascular endothelial growth factor (VEGF) expression, and vascularization/angiogenesis as well as decreased CD68+ cell accumulation. However, cell differentiation and VEGF expression were not demonstrated in deposited cells. The results suggest that the promotion of short-term functional recovery in sciatic nerve crush injury by G-CSF involves a paracrine modulatory effect and a bone marrow-derived CD34+ cell mobilizing effect.     

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.     

Neural Stem Cells Enhance Nerve Regeneration after Sciatic Nerve Injury in Rats

With the development of tissue engineering and the shortage of autologous nerve grafts in nerve reconstruction, cell transplantation in a conduit is an alternative strategy to improve nerve regeneration. The present study evaluated the effects and mechanism of brain-derived neural stem cells (NSCs) on sciatic nerve injury in rats. At the transection of the sciatic nerve, a 10-mm gap between the nerve stumps was bridged with a silicon conduit filled with 5?×?10⁵ NSCs. In control experiments, the conduit was filled with nerve growth factor (NGF) or normal saline (NS). The functional and morphological properties of regenerated nerves were investigated, and expression of hepatocyte growth factor (HGF) and NGF was measured. One week later, there was no connection through the conduit. Four or eight weeks later, fibrous connections were evident between the proximal and distal segments. Motor function was revealed by measurement of the sciatic functional index (SFI) and sciatic nerve conduction velocity (NCV). Functional recovery in the NSC and NGF groups was significantly more advanced than that in the NS group. NSCs showed significant improvement in axon myelination of the regenerated nerves. Expression of NGF and HGF in the injured sciatic nerve was significantly lower in the NS group than in the NSCs and NGF groups. These results and other advantages of NSCs, such as ease of harvest and relative abundance, suggest that NSCs could be used clinically to enhance peripheral nerve repair.     

Extracellular vesicles from human umbilical cord mesenchymal stem cells improve nerve regeneration after sciatic nerve transection in rats

Peripheral nerve injury results in limited nerve regeneration and severe functional impairment. Mesenchymal stem cells (MSCs) are a remarkable tool for peripheral nerve regeneration. The involvement of human umbilical cord MSC‐derived extracellular vesicles (hUCMSC‐EVs) in peripheral nerve regeneration, however, remains unknown. In this study, we evaluated functional recovery and nerve regeneration in rats that received hUCMSC‐EV treatment after nerve transection. We observed that hUCMSC‐EV treatment promoted the recovery of motor function and the regeneration of axons; increased the sciatic functional index; resulted in the generation of numerous axons and of several Schwann cells that surrounded individual axons; and attenuated the atrophy of the gastrocnemius muscle. hUCMSC‐EVs aggregated to rat nerve defects, down‐regulated interleukin (IL)‐6 and IL‐1β, up‐regulated IL‐10 and modulated inflammation in the injured nerve. These effects likely contributed to the promotion of nerve regeneration. Our findings indicate that hUCMSC‐EVs can improve functional recovery and nerve regeneration by providing a favourable microenvironment for nerve regeneration. Thus, hUCMSC‐EVs have considerable potential for application in the treatment of peripheral nerve injury.     

Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat

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.     

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.     

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.     

Recovery of CNS Pathway Innervating the Sciatic Nerve Following Transplantation of Human Neural Stem Cells in Rat Spinal Cord Injury

Stem cell research has been attained a greater attention in most fields of medicine due to its potential for many incurable diseases through replacing or helping the regeneration of damaged cells or tissues. Here, we demonstrated the functional recovery and structural connection of the central nervous system pathway innervating the sciatic nerve after total transection of the spinal cord followed by the transplantation of human neural stem cells (hNSC) in the injured rat spinal cord site. The limb function of hNSC-treated group recovered dramatically compared with that in the sham group by Basso–Beattie–Bresnahan (BBB) scores. Transplanted hNSC differentiated into astrocytes and neurons in the injured site. In addition, immunohistochemistry for growth-associated protein 43 showed axonal regeneration in the injured spinal cord site. The pseudorabies viral-Ba (PRV-Ba) tracing method revealed that transplanted hNSC and their differentiated neurons showed positive labeling after sciatic nerve injection. In addition, the PRV-Ba labeling was also observed in several nuclei in the brain innervating the sciatic nerve. This result implies that the rat CNS motor pathway could be reconstructed by hNSC transplantation, and it may contribute to the functional recovery of the limb.     

Down‐regulation of long non‐coding RNA MEG3 promotes Schwann cell proliferation and migration and repairs sciatic nerve injury in rats

Peripheral nerve injury and regeneration are complex processes and involve multiple molecular and signalling components. However, the involvement of long non‐coding RNA (lncRNA) in this process is not fully clarified. In this study, we evaluated the expression of the lncRNA maternally expressed gene 3 (MEG3) in rats after sciatic nerve transection and explored its potential mechanisms. The expression of lncRNA MEG3 was up‐regulated following sciatic nerve injury and observed in Schwann cells (SCs). The down‐regulation of lncRNA MEG3 in SCs enhanced the proliferation and migration of SCs via the PTEN/PI3K/AKT pathway. The silencing of lncRNA MEG3 promoted the migration of SCs and axon outgrowth in rats after sciatic nerve transection and facilitated rat nerve regeneration and functional recovery. Our findings indicated that lncRNA MEG3 may be involved in nerve injury and injured nerve regeneration in rats with sciatic nerve defects by regulating the proliferation and migration of SCs. This gene may provide a potential therapeutic target for improving peripheral nerve injury.     

Brain-Derived Neurotrophic Factor from Bone Marrow-Derived Cells Promotes Post-Injury Repair of Peripheral Nerve

Brain-derived neurotrophic factor (BDNF) stimulates peripheral nerve regeneration. However, the origin of BNDF and its precise effect on nerve repair have not been clarified. In this study, we examined the role of BDNF from bone marrow-derived cells (BMDCs) in post-injury nerve repair. Control and heterozygote BDNF knockout mice (BDNF+/−) received a left sciatic nerve crush using a cerebral blood clip. Especially, for the evaluation of BDNF from BMDCs, studies with bone marrow transplantation (BMT) were performed before the injury. We evaluated nerve function using a rotarod test, sciatic function index (SFI), and motor nerve conduction velocity (MNCV) simultaneously with histological nerve analyses by immunohistochemistry before and after the nerve injury until 8 weeks. BDNF production was examined by immunohistochemistry and mRNA analyses. After the nerve crush, the controls showed severe nerve dysfunction evaluated at 1 week. However, nerve function was gradually restored and reached normal levels by 8 weeks. By immunohistochemistry, BDNF expression was very faint before injury, but was dramatically increased after injury at 1 week in the distal segment from the crush site. BDNF expression was mainly co-localized with CD45 in BMDCs, which was further confirmed by the appearance of GFP-positive cells in the BMT study. Variant analysis of BDNF mRNA also confirmed this finding. BDNF+/− mice showed a loss of function with delayed histological recovery and BDNF+/+→BDNF+/− BMT mice showed complete recovery both functionally and histologically. These results suggested that the attenuated recovery of the BDNF+/− mice was rescued by the transplantation of BMCs and that BDNF from BMDCs has an essential role in nerve repair.     

Walking track analysis: a long-term assessment of peripheral nerve recovery.

Functional recovery following sciatic, tibial, and peroneal nerve injury was assessed over a 1-year period using walking track analysis in the rat. Internal neurolysis did not affect nerve function. Crush injury induced a temporary, but complete, loss of function that recovered to control levels by 4 weeks. Nerve transection resulted in complete loss of function without any evidence of recovery. After nerve repair, functional recovery occurred, reaching near-optimal recovery by 12 weeks. The degree of functional recovery varied with the specific nerve involved. The sciatic nerve recovered 41 percent of function, whereas the tibial nerve recovered 54 percent of function. The peroneal nerve exhibited the highest degree of recovery, achieving functional levels similar to control values. Assessment of neural regeneration using walking track analysis appears to be a valuable addition to the traditional methods of histology and electrophysiology.     

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     

N-Terminal Arginylation of Sciatic Nerve and Brain Proteins Following Injury

N-terminal protein arginylation has been demonstrated in vitro and in situ and has been reported to increase following injury to sciatic nerves of rats. The present study attempts to demonstrate these reactions in vivo by applying [³H]Arg to the cut end of sciatic nerves in anesthetized rats and assaying for N-terminal arginylation using Edman chemistry and acid precipitation of labeled proteins in the proximal nerve segment. No evidence was found for arginylation in an aqueous soluble fraction. However, N-terminal arginylation was detected in a urea soluble fraction at 2 hours after nerve crush. The data show that arginylation of rat sciatic nerve proteins occurs in vivo and suggest that the arginylated proteins formed an aqueous insoluble/urea soluble aggregate after arginylation. In other experiments, rat brains were injured and assayed for arginylation in vitro to test the hypothesis that injury causes an up-regulation of these reactions. Results showed an activation of the reaction at 2 hours post crush and indicate that increases in N-terminal arginylation are likely to be a general response to injury in nervous tissue.