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We determined if combined administration of JNK-inhibitors and HGF (hepatocyte-growth-factor) would restore erectile-function through both antiapoptotic and regenerative effects in a rat model of cavernous-nerve-crush-injury (CNCI), and compared the results with administration of JNK-inhibitor alone or HGF alone. We randomized 70 rats into 5 groups: sham-surgery-group (S), CNCI (I) group, a group treated with once-daily intraperitoneal-administration of 10.0-mg/kg of JNK-inhibitors (J), a twice-weekly intracavernosal-administration of 4.2-μg HGF group (H), and a combined-treatment with 10.0-mg/kg JNK-inhibitors and 4.2-μg HGF group (J+H). We investigated erectile-responses to electrostimulation, histological-staining, caspase-3-activity-assay, and immunoblotting at two-weeks postoperatively. The three treatment groups showed improvements in erectile-responses (ICP/MAP and AUC/MAP ratios) compared to Group-I. The erectile-responses in Group-J+H were greater than those in Group-J or Group-H. The erectile-responses in Group-J+H were generally normalized. Caspase-3-activity and cJun-phosphorylation in Group-J and Group-J+H improved compared to Group-I, whereas caspase-3-activity in Group-H partially improved. Protein-expression of PECAM-1, eNOS-phosphorylation, and smooth-muscle content in Group-J+H were normalized, although those in Group-J or Group-H were partially restored. Combination therapy with JNK-inhibitors and HGF can generally normalize erectile-function through anti-apoptosis and preservation of endothelium or SM in rat CNCI model. The combined treatment appears to be superior to the respective agent alone in terms of therapeutic effects.

Peripheral nerve injuries can significantly reduce quality of life. While some recover, most do not recover fully, resulting in neuropathic pain and loss of sensation and motor function. Research on the mechanisms of peripheral nerve regeneration could elucidate poor patient outcomes and potential treatments. This study was designed to determine if brain derived neurotrophic factor (BDNF) is necessary for pudendal nerve regeneration and functional recovery. Peripheral administration of tyrosine kinase B functional chimera (TrkB) was used to inhibit the BDNF regenerative pathway. Female Sprague-Dawley rats received tyrosine kinase B functional chimera (TrkB) or saline after a pudendal nerve crush (PNC) or Sham PNC and were divided into three groups: Sham PNC, PNC + Saline, and PNC + TrkB. Seven days after injury, relative β II tubulin expression (1.0 ± 0.2) was significantly decreased after PNC + TrkB compared to PNC + saline (2.9 ± 1.0). Three weeks after injury, BDNF plasma concentration (1320.8 ± 278.1 pg/ml) was significantly reduced in PNC + TrkB compared to PNC + saline rats (2053.4 ± 211.0 pg/ml). Pudendal nerve motor branch firing rate (54.0 ± 9.5 Hz) was significantly decreased in the PNC + TrkB group compared to the PNC + saline group (120.4 ± 17.1 Hz); while nerve firing rate of the PNC + saline group was not significantly different from sham PNC rats (121.8 ± 26.6 Hz). This study demonstrated that peripheral administration of TrkB bound free BDNF and inhibited the regenerative response after PNC. BDNF is necessary for normal PN motor branch recovery after PNC., (Published by Elsevier Inc.)

Traumatic injury to the peripheral nervous system (PNS) is the most common cause of acquired nerve damage and impairs the quality of life of patients. The success of nerve regeneration depends on distal stump degeneration, tissue clearance and remodeling, processes in which the immune system participates. We previously reported improved motor recovery in sciatic nerve crush mice following adoptive transfer of lymphocytes, which migrated to the lesion site. However, lymphocyte activity and the nerve tissue response remain unexplored. Thus, in the present study, we evaluated sciatic nerve regeneration and T cell polarization in lymphocyte recipient mice. Splenic lymphocytes were isolated from mice 14 days after sciatic nerve crush and transferred to axotomized animals three days postinjury. Immediate lymphocyte migration to the crushed nerve was confirmed by in vivo imaging. Phenotyping of T helper (Th) cells by flow cytometry revealed an increased frequency of the proinflammatory Th1 and Th17 cell subsets in recipient mice at 7 days and showed that the frequency of these cells remained unchanged for up to 21 days. Moreover, nerve regeneration was improved upon cell therapy, as shown by sustained immunolabeling of axons, Schwann cells, growth-associated protein 43 and BDNF from 14 to 28 days after lesion. Macrophage and IgG immunolabeling were also higher in cell-transferred mice at 14 and 21 days following nerve crush. Functionally, we observed better sensory recovery in the lymphocyte-treated group. Overall, our data demonstrate that enhanced inflammation early after nerve injury has beneficial effects for the regenerative process, improving tissue clearance and axonal regrowth towards the target organs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

VGF nerve growth factor inducible (VGF) is a polypeptide that is induced by neurotrophic factors and is involved in neurite growth and neuroprotection. The mRNA of the Vgf gene has been detected in the adult rat retina, however the roles played by VGF in the retina are still undetermined. Thus, the purpose of this study was to determine the effects of VGF on the retinal ganglion cells (RGCs) of mice in the optic nerve crush (ONC) model, rat-derived primary cultured RGCs and human induced pluripotent stem cells (iPSCs)-derived RGCs. The mRNA and protein of Vgf were upregulated after the ONC. Immunostaining showed that the VGF was located in glial cells including Müller glia and astrocytes but not in the retinal neurons and their axons. AQEE-30, a VGF peptide, suppressed the loss of RGCs induced by the ONC, and it increased survival rat-derived RGCs and promoted the outgrowth of neurites of rat and human iPSCs derived RGCs in vitro. These findings indicate that VGF plays important roles in neuronal degeneration and has protective effects against the ONC on RGCs. Thus, VGF should be considered as a treatment of RGCs degeneration.

Urinary incontinence affects 40% of elderly men, is common in diabetic patients and in men treated for prostate cancer, with a prevalence of up to 44%. Seventy-two percent of prostatectomy patients develop stress urinary incontinence (SUI) in the first week after surgery and individuals who do not recover within 6 months generally do no regain function without intervention. Incontinence has a profound impact on patient quality of life and a critical unmet need exists to develop novel and less invasive SUI treatments. During prostatectomy, the cavernous nerve (CN), which provides innervation to the penis, undergoes crush, tension, and resection injury, resulting in downstream penile remodeling and erectile dysfunction in up to 85% of patients. There are other nerves that form part of the major pelvic ganglion (MPG), including the hypogastric (HYG, sympathetic) and pelvic (PN, parasympathetic) nerves, which provide innervation to the bladder and urethra. We examine if HYG and PNs are injured during prostatectomy contributing to SUI, and if Sonic hedgehog (SHH) regulatory mechanisms are active in the PN and HYG nerves. CN, PN, HYG and ancillary (ANC) of uninjured, sham and CN crush/MPG tension injured (prostatectomy model) adult Sprague Dawley rats (n = 37) were examined for apoptosis, sonic hedgehog (SHH) pathway, and intrinsic and extrinsic apoptotic mechanisms. Fluorogold tracing from the urethra/bladder was performed. PN and HYG response to SHH protein was examined in organ culture. TUNEL, immunohistochemical analysis for caspase-3 cleaved, -8, -9, SHH, Patched and Smoothened (SHH receptors), and neurite formation, were examined. Florogold positive neurons in the MPG were reduced with CN crush. Apoptosis increased in glial cells of the PN and HYG after CN crush. Caspase 9 was abundant in glial cells (intrinsic), while caspase-8 was not observed. SHH and its receptors were abundant in neurons and glia of the PN and HYG. SHH treatment increased neurite formation. PN and HYG injury occur concomitant with CN injury during prostatectomy, likely contributing to SUI. PN and HYG response to SHH treatment indicates an avenue for intervention to promote regeneration and prevent SUI.

Pim-1 is a proto-oncogene which has been discovered to involve in cell proliferation, differentiation, and survival. In this study, we observed the expression of Pim-1 in neonatal and adult rat retina and the changes in rat retina following optic nerve crush (ONC) in order to explore the relationship between Pim-1 and the survival of retinal ganglion cells (RGC). We discovered that Pim-1 was distributed mainly in retinal pigment epithelial cells (RPE) and retinal ganglion cell layer (GCL) in normal newborn rats, and it appeared in RPE, cone rod cell layer and GCL in normal adult rats by immunohistochemistry. Our double immunofluorescent staining of Pim-1 and γ-synuclein further confirmed that Pim-1 was localized in 80% of RGC. Moreover, we found that the amount of Pim-1 mRNA and protein in adult rat retina was transiently increased after ONC and then decreased 2 weeks after ONC, and the expression level was lower than that of neonatal rat retina under all conditions. We also discovered that Pim-1 expression in GCL detected by immunohistochemistry was upregulated at Day 1 and Day 3 after ONC, but downregulated at Day 14 after ONC when the survival of RGC was decreased and the apoptotic cells in GCL were increased by hematoxylin-eosin staining, immunohistochemistry, and TUNEL detection. We suggest that the overexpression of Pim-1 in the RGC is related to the optic nerve repair while the low expression of Pim-1 in RGC may be associated with apoptosis and weak intrinsic regeneration ability of RGC. Anat Rec, 301:1968-1976, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

The regenerative capacity of CNS tracts has ever been a great hurdle to regenerative medicine. Although recent studies have described strategies to stimulate retinal ganglion cells (RGCs) to regenerate axons through the optic nerve, it still remains to be elucidated how these therapies modulate the inhibitory environment of CNS. Thus, the present work investigated the environmental content of the repulsive axon guidance cues, such as Sema3D and its receptors, myelin debris, and astrogliosis, within the regenerating optic nerve of mice submitted to intraocular inflammation + cAMP combined to conditional deletion of PTEN in RGC after optic nerve crush. We show here that treatment was able to promote axonal regeneration through the optic nerve and reach visual targets at twelve weeks after injury. The Regenerating group presented reduced MBP levels, increased microglia/macrophage number, and reduced astrocyte reactivity and CSPG content following optic nerve injury. In addition, Sema3D content and its receptors are reduced in the Regenerating group. Together, our results provide, for the first time, evidence that several regenerative repulsive signals are reduced in regenerating optic nerve fibers following a combined therapy. Therefore, the treatment used made the CNS microenvironment more permissive to regeneration.

Recently, we showed that matrix metalloproteinase-12 (MMP-12) is highly expressed in microglia and myeloid infiltrates, which are presumably involved in blood⁻brain barrier (BBB) leakage and subsequent neuronal cell death that follows status epilepticus (SE). Here, we assessed the effects of a hydroxypyrone-based inhibitor selective for MMP-12 in the pilocarpine-induced SE rat model to determine hippocampal cell survival. In the hippocampus of rats treated with pilocarpine, intra-hippocampal injections of the MMP-12 inhibitor protected Cornu Ammonis 3 (CA3) and hilus of dentate gyrus neurons against cell death and limited the development of the ischemic-like lesion that typically develops in the CA3 stratum lacunosum-moleculare of the hippocampus. Furthermore, we showed that MMP-12 inhibition limited immunoglobulin G and albumin extravasation after SE, suggesting a reduction in BBB leakage. Finally, to rule out any possible involvement of seizure modulation in the neuroprotective effects of MMP-12 inhibition, neuroprotection was also observed in the retina of treated animals after optic nerve crush. Overall, these results support the hypothesis that MMP-12 inhibition can directly counteract neuronal cell death and that the specific hydroxypyrone-based inhibitor used in this study could be a potential therapeutic agent against neurological diseases/disorders characterized by an important inflammatory response and/or neuronal cell loss.

Injury to the peripheral nervous system begins a well-characterized process within both neurons and Schwann cells to allow axonal regrowth, remyelination, and functional repair. Models of peripheral nerve injury have been widely used to study the behavior of Schwann cells, neurons, and other cell types such as macrophages as the events of Wallerian degeneration and regeneration take place. The most commonly used approaches in rodent models to model nerve injury in human patients are sciatic nerve transection and nerve crush, and both have well established time courses of demyelination, immune cell influx, axonal regrowth, and remyelination. We describe the techniques of sciatic nerve surgery for transection and crush injury, together with methods for the analysis of events within peripheral nerve repair in these two models.

The effects of sciatic nerve crush (SNC) and treatment with Riluzole on muscle activity during unrestrained locomotion were identified in an animal model by analysis of the EMG activity recorded from soleus (Sol) and extensor digitorum longus (EDL) muscles of both hindlimbs; in intact rats (IN) and in groups of rats treated for 14 days with saline (S) or Riluzole (R) after right limb nerve crush at the 1st (1S and 1R) or 2nd (2S and 2R) day after birth. Changes in the locomotor pattern of EMG activity were correlated with the numbers of survived motor units (MUs) identified in investigated muscles. S rats with 2-8 and 10-28 MUs that survived in Sol and EDL muscles respectively showed increases in the duration and duty factor of muscle EMG activity and a loss of correlation between the duty factors of muscle activity, and abnormal flexor-extensor co-activation 3 months after SNC. R rats with 5, 6 (Sol) and 15-29 MUs (EDL) developed almost normal EMG activity of both Sol and control EDL muscles, whereas EDL muscles with SNC showed a lack of recovery. R rats with 8 (Sol) and 23-33 (EDL) MUs developed almost normal EMG activities of all four muscles. A subgroup of S rats with a lack of recovery and R rats with almost complete recovery that had similar number of MUs (8 and 24-28 vs 8 and 23-26), showed that the number of MUs was not the only determinant of treatment effectiveness. The results demonstrated that rats with SNC failed to develop normal muscle activity due to malfunction of neuronal circuits attenuating EDL muscle activity during the stance phase, whereas treatment with Riluzole enabled almost normal EMG activity of Sol and EDL muscles during locomotor movement., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Cavernous nerve injury (CNI) in rats and radical prostatectomy (RP) in men result in loss of nitrergic function and increased adrenergic-neurogenic contractions of cavernosal tissue., Aim: To evaluate the modulation of the α-adrenergic system as a strategy to relieve erectile dysfunction (ED) and functional cavernosal alterations induced by CNI., Methods: A non-selective α-blocker (phentolamine 1 mg/kg daily), a selective α 1A -blocker (silodosin [SILOD] 0.1 mg/kg daily), or vehicle was orally administered for 4 weeks after bilateral crush CNI (BCNI). Erectile and neurogenic responses of the corpus cavernosum (CC) were evaluated. The acute effects of SILOD also were evaluated in vivo (0.03 mg/kg intravenously) and ex vivo (10 nmol/L). The effects of SILOD and tadalafil (TAD) on nitrergic relaxations were determined in human CC from patients with ED with a vascular etiology or ED secondary to RP., Main Outcome Measures: Erectile responses in vivo in rats and neurogenic contractions and relaxations of rat and human CC., Results: Long-term treatment with SILOD significantly improved erectile responses and allowed for the potentiation of erectile responses by acute treatment with TAD (0.3 mg/kg intravenously) in rats with BCNI. SILOD partly recovered nitrergic relaxations and normalized neurogenic contractions in CC from rats with BCNI. Long-term treatment with SILOD partly prevented BCNI-induced decreases in neuronal nitric oxide synthase expression. Acute administration of SILOD (0.03 mg/kg intravenously) improved erectile responses in vivo and potentiated nitrergic relaxation and decreased neurogenic contractions ex vivo in CC from rats with BCNI. In human CC from patients with ED with a vascular etiology, TAD (30 nmol/L), SILOD (10 nmol/L), or their combination increased nitrergic relaxations. Potentiation by TAD was lost in human CC from patients with ED after RP but was recovered after co-treatment with SILOD., Conclusion: α-Adrenergic modulation, especially selective α 1A -blockade, improves erectile and cavernosal functions after BCNI. Modulation of the adrenergic system, mainly in combination strategies, could have a role in the management of ED after RP., (Copyright © 2016 International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.)

Little is known about the reasons for occurrence of facial nerve palsy after removal of cerebellopontine angle tumors. Since the intra-arachnoidal portion of the facial nerve is considered to be so vulnerable that even the slightest tension or pinch may result in ruptured axons, we tested whether a graded stretch or controlled crush would affect the postoperative motor performance of the facial (vibrissal) muscle in rats. Thirty Wistar rats, divided into five groups (one with intact controls and four with facial nerve lesions), were used. Under inhalation anesthesia, the occipital squama was opened, the cerebellum gently retracted to the left, and the intra-arachnoidal segment of the right facial nerve exposed. A mechanical displacement of the brainstem with 1 or 3 mm toward the midline or an electromagnet-controlled crush of the facial nerve with a tweezers at a closure velocity of 50 and 100 mm/s was applied. On the next day, whisking motor performance was determined by video-based motion analysis. Even the larger (with 3 mm) mechanical displacement of the brainstem had no harmful effect: The amplitude of the vibrissal whisks was in the normal range of 50°-60°. On the other hand, even the light nerve crush (50 mm/s) injured the facial nerve and resulted in paralyzed vibrissal muscles (amplitude of 10°-15°). We conclude that, contrary to the generally acknowledged assumptions, it is the nerve crush but not the displacement-induced stretching of the intra-arachnoidal facial trunk that promotes facial palsy after cerebellopontine angle surgery in rats.

Aims: Histamine and serotonin-related pharmaceuticals have the potential to modulate micturition and continence. The aim of this study was to determine if treatment with histamine and/or serotonin improves stress urinary incontinence (SUI) in female rats., Methods: Twenty-six age-matched female rats underwent pudendal nerve crush and vaginal distension (PNC + VD), to produce SUI. One week after injury, rats were treated subcutaneously with saline, histamine (1.1 μg), serotonin (2μg), or the combination of both twice daily for another week. A sham injured group received sham PNC + VD and were treated with saline (n = 7). Leak point pressure (LPP) testing with simultaneous external urethral sphincter (EUS) electromyography (EMG) was conducted 2 weeks after injury. The urethra was harvested for qualitative and quantitative histology. Data were analyzed with a one-way ANOVA and Student-Newman-Keuls posthoc test with P < 0.05 indicating statistically significant differences between groups., Results: Combination treatment significantly increased LPP after PNC + VD compared to injured sham treatment and treatment with either histamine or serotonin alone. Compared to injured sham treated rats, all three treatments significantly increased EUS EMG amplitude at both baseline and peak pressure and EUS EMG firing rate at peak pressure during LPP testing. There were more consistent urethral striated muscle fibers and thicker smooth and striated muscle with combination and histamine treatment. There was a statistically significant shift to a greater proportion of thicker collagen fibers in the urethra in serotonin and combination treated rats compared with injured sham treated rats., Conclusions: Combination treatment was the most effective and may provide an effective therapy for SUI. Neurourol. Urodynam. 35:703-710, 2016. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

Background Intimal injury plays a critical role in initiating the pathogenesis of thrombosis formation after microsurgical anastomosis. Application of stromal cell-derived factor-1α (SDF-1α) is reported to promote early regeneration of injured intima through migration of endothelial cells and mobilization of endothelial progenitor cells. We therefore hypothesized that local transfer of SDF-1α gene would inhibit microsurgical anastomotic thrombosis. Methods Sixty Sprague-Dawley rats were used and divided randomly into three groups (SDF-1α group, plasmid group, and saline group) in this study. Plasmid DNA encoding SDF-1α, empty plasmid, and saline were injected into the left femoral muscles of rats from each group, respectively. Seven days after injection, peripheral blood samples were obtained to measure the plasma levels of SDF-1α and nitric oxide (NO). The left femoral artery of each rat was crushed, transected, and repaired by end-to-end microsurgical anastomosis. Vascular patency was assessed at 15, 30, and 120 minutes after reperfusion using milk test. Thrombosis formation was assessed with hematoxylin and eosin staining and scanning electron microscopy at 120 minutes postoperatively. Results The plasma levels of SDF-1α and NO in SDF-1α group were significantly higher than those in plasmid group and saline group (p < 0.01). The patency rate in SDF-1α group was significantly higher than that in control groups at 120 minutes after reperfusion (p < 0.05). Treatment of SDF-1α significantly reduced the size of thrombotic occlusion when compared with controls (p < 0.05). All SDF-1α recipients exhibited decreased thrombosis under scanning electron microscopy. Conclusions The current study demonstrated that local transfer of SDF-1α gene increases arterial patency and inhibits microsurgical anastomotic thrombosis in a crush model of femoral artery in rat. The antithrombotic effect of SDF-1α may be mediated through increased production of endogenous NO. These findings provide a novel approach for inhibition of microsurgical anastomotic thrombosis., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

The aim of the present study was to use a model of simulated human childbirth in rats to determine the damage to genitourinary structures and behavioral signs of urinary dysfunction induced by vaginal distension (VD) in female rats. In experiment 1, the length of the genitourinary tract and the nerves associated with it were measured immediately after simulated human delivery induced by VD or sham (SH) procedures. Electroneurograms of the dorsal nerve of the clitoris (DNC) were also recorded. In experiment 2, histological characteristics of the bladder and major pelvic ganglion of VD and SH rats were evaluated. In experiment 3, urinary parameters were determined in conscious animals during 6 h of dark and 6 h of light before and 3 days after VD or SH procedures. VD significantly increased distal vagina width (P < 0.001) and the length of the motor branch of the sacral plexus (P < 0.05), DNC (P < 0.05), and vesical nerves (P < 0.01) and decreased DNC frequency and amplitude of firing. VD occluded the pelvic urethra, inducing urinary retention, hematomas in the bladder, and thinness of the epithelial (P < 0.05) and detrusor (P < 0.01) layers of the bladder. Major pelvic ganglion parameters were not modified after VD. Rats dripped urine in unusual places to void, without the stereotyped behavior of micturition after VD. The neuroanatomic injuries after VD occur alongside behavioral signs of urinary incontinence as determined by a new behavioral tool for assessing micturition in conscious animals.

In cranial and spinal nerve ganglia, both axotomized primary sensory neurons without regeneration (axotomy-nonregenerative neurons) and spared intact primary sensory neurons adjacent to axotomized neurons (axotomy-spared neurons) have been definitely shown to participate in pain transmission in peripheral neuropathic pain states. However, whether axotomized primary sensory neurons with regeneration (axotomy-regenerative neurons) would be integral components of neural circuits underlying peripheral neuropathic pain states remains controversial. In the present study, we utilized an adult rat sciatic nerve crush model to systematically analyze pain behaviors on the glabrous plantar surface of the hindpaw sural nerve skin territories. To the best of our knowledge, our results for the first time showed that heat hyperalgesia, cold allodynia, mechanical allodynia, and mechanical hyperalgesia emerged and persisted on the glabrous sural nerve skin areas after adult rat sciatic nerve crush. Interestingly, mechanical hyperalgesia was sexually dimorphic. Moreover, with our optimized immunofluorescence staining protocol of free-floating thick skin sections for wide-field epifluorescence microscopic imaging, changes in purely regenerative reinnervation on the same skin areas by axotomized primary sensory afferents were shown to be paralleled by those pathological pain behaviors. To our surprise, Protein Gene Product 9.5-immunoreactive nerve fibers with regular and large varicosities ectopically emigrated into the upper dermis of the glabrous sural nerve skin territories after adult rat sciatic nerve crush. Our results indicated that axotomy-regenerative primary sensory neurons could be critical elements in neural circuits underlying peripheral neuropathic pain states. Besides, our results implied that peripheral neuropathic pain transmitted by axotomy-regenerative primary sensory neurons alone might be a new dimension in the clinical therapy of peripheral nerve trauma beyond regeneration.

Purpose: Optic nerve crush (ONC) and subsequent axonal damage can be used in rodents to study the mechanism of retinal ganglion cell (RGC) degeneration. Here, we examined electroretinograms (ERGs) in post-ONC mice to investigate changes in the positive scotopic threshold response (pSTR). We then compared these changes with molecular and morphological changes to identify early objective biomarkers of RGC dysfunction., Methods: Fifty 12-week-old C57BL/6 mice were included. ONC was used to induce axonal injury in the right eye of each animal, with the left eye used as a control. The expression of the RGC markers Brn3a and Brn3b was measured on days 1, 2, 3, 5 and 7 after ONC with quantitative real-time PCR. ERGs were recorded under dark adaptation with the stimulus intensity increasing from -6.2 to 0.43 log cd-s/m(2) on days 1, 2, 3, 5, 7 and 10 after ONC. The pSTR, a- and b-wave amplitudes were measured. Inner retinal thickness around the optic nerve head was measured with spectral-domain optical coherence tomography on days 0, 2, 5, 7 and 10 after ONC., Results: The expression of Brn3a and Brn3b began to significantly decrease on day 1 and day 2, respectively (P < 0.01). The amplitude of the pSTR underwent rapid, significant deterioration on day 3, after which it fell gradually (P < 0.01), while the a- and b-wave amplitudes remained unchanged throughout the experiment. Inner retinal thickness gradually decreased, with the most significant reduction on day 10 (P < 0.01)., Conclusions: Decrease in pSTR likely reflected the early loss of RGC function after ONC and that declining expression of RGC-specific genes preceded anatomical and functional changes in the RGCs.

This study evaluated the effect of low-level laser therapy (LLLT; 15 J/cm(2)) and a latex protein (F1) on a crush injury of the sciatic (ischiadicus) nerve. Seventy-two rats (male, 250 g) were divided into 6 groups: CG, control; EG, exposed nerve; IG, injured nerve without treatment; LG, injured nerve with LLLT; HG, injured nerve with F1; and LHG, injured nerve with LLLT and F1. After 4 or 8 weeks, the animals were euthanized and samples of the sciatic nerve were collected for morphometric and high-resolution scanning electron microscopy (HRSEM) analysis. After 4 weeks, the morphometry revealed improvements in the treated animals, and the HG appeared to be the most similar to the CG; after 8 weeks, the injured groups showed improvements compared to the previous period, and the results of the treatment groups were more similar to one another. At HRSEM after 4 weeks, the treated groups were similar and showed improvement compared to the IG; after 8 weeks, the LHG and HG had the best results. In conclusion, the treatments resulted in improvement after the nerve injury, and this recovery was time-dependent. In addition, the use of the F1 resulted in the best morphometric and ultrastructural findings., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The purpose of this study was to determine the temporal and spatial changes in the expression of AQP4 and AQP9 in the optic nerve after it is crushed. The left optic nerves of rats were either crushed (crushed group) or sham operated (sham group), and they were excised before, and at 1, 2, 4, 7, and 14 days later. Four optic nerves were pooled for each time point in both groups. The expression of AQP4 and AQP9 was determined by western blot analyses. Immunohistochemistry was used to determine the spatial expression of AQP4, AQP9, and GFAP in the optic nerve. Optic nerve edema was determined by measuring the water content in the optic nerve. The barrier function of the optic nerve vessels was determined by the extravasated Evans blue dye on days 7 and 14. The results showed that the expression of AQP4 was increased on day 1 but the level was significantly lower than that in the sham group on days 4 and 7 (P<0.05). In contrast, the expression of AQP9 gradually increased, and the level was significantly higher than that in the sham group on days 7 and 14 (P<0.05, Tukey-Kramer). The down-regulation of AQP4 was associated with crush-induced optic nerve edema, and the water content of the nerve was significantly increased by 4.3% in the crushed optic nerve from that of the untouched fellow nerve on day 7. The expression of AQP4 and GFAP was reduced at the crushed site where AQP4-negative and AQP9-positive astrocytes were present. The barrier function was impaired at the crushed site on days 7 and 14, restrictedly where AQP4-negative and AQP9-positive astrocytes were present. The presence of AQP9-positive astrocytes at the crushed site may counteract the metabolic damage but this change did not fully compensate for the barrier function defect.

GTP-binding protein Gem, a member protein of the Ras superfamily, can regulate actin cytoskeleton reorganization mediated by Rho-associated coiled-coil-containing protein kinase (ROCK). One attractive activity of the ROCK is playing a potential role in physiological and pathological process in retinal ganglion cells (RGCs) apoptosis. However, the function of Gem in retina is still with limited understanding. To investigate whether Gem is involved in optic nerve injury, we performed an optic nerve crush (ONC) model in adult rats. Western blot analysis indicated that Gem was significantly increased in the retina at the 3rd day after ONC. Meanwhile, double-immunofluorescent staining showed that Gem expression was mainly up-regulated in ganglion cell layer and co-localized with NeuN (a marker of RGCs). Additionally, the co-localizations of Gem/active-caspase-3 and Gem/TUNEL-positive cells were detected in RGCs. Furthermore, the expression of active-caspase-3 and TUNEL-positive cells was parallel with that of Gem. Finally, expression pattern of ROCK family (only ROCK2 but not ROCK1) was increased in the differentiated process, which was collected with the expression of GEM and active-caspase-3. Based on the present results, it is suggested that Gem might play a crucial role in RGCs apoptosis after ONC, which might be involved in ROCK pathway.

Purpose: We investigated the progressive nature of neurodegenerative structural changes following injury to retinal ganglion cell (RGC) axons using quantifiable and noninvasive in vivo imaging techniques., Methods: To track degenerative RGC progression in retinas following optic nerve crush (ONC) injury, spectral-domain optical coherence tomography (SD-OCT) was used to quantitate the RGC nerve fiber layer (NFL) density. The RGC soma cell density (RCD) was measured by confocal scanning laser ophthalmoscopy (CSLO). The RCD counts were performed using blood vessels as landmarks to anatomically track defined progressive changes in enhanced yellow fluorescent fusion protein (EYFP)-labeled RGCs., Results: Following ONC injury, 68% of the observed decrease in RCD measured by CSLO and 54% of the NFL thickness obtained by SD-OCT imaging (N=4 retinas) occurred within the first week. Between days 7 and 14, an additional 22% decrease in RCD was concurrent with a 31% decrease in overall NFL thickness. Finally, between days 14 and 21, an additional 10% decrease in RCD measured in vivo by CSLO and 15% decrease in NFL thickness by SD-OCT was observed., Conclusions: Our data suggest that in vivo CSLO imaging of EYFP-RGC expression and SD-OCT measured NFL thickness are fast and reliable methods that longitudinally track neurodegenerative progression following ONC injury. Neurodegenerative changes in NFL thickness measured by SD-OCT imaging have the same overall trajectory as those observed by CSLO for RCD; however, changes in NFL thickness initially lag behind in vivo RGC soma counts with a slower decline in overall measurable change., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: We characterized the morphologic and functional changes in optic nerve crushed mice and evaluated electroretinogram (ERG) responses as tools to monitor retinal ganglion cell (RGC) dysfunction., Methods: We performed optic nerve crush (ONC) unilaterally in adult BALB/cJ mice. The neuronal loss in the RGC layer (GCL) and superior colliculus (SC) was determined by Nissl staining. Retinal thickness was assessed by spectral-domain optical coherence tomography (SD-OCT) imaging. Retinal function was determined by pattern ERG and full-field flash ERG. Responses of pattern ERG, positive scotopic threshold response (pSTR), scotopic oscillatory potentials (OPs), and photopic negative response (PhNR) were analyzed., Results: The ONC induced progressive neuronal loss in GCL and contralateral SC starting from 7 and 28 days following ONC, respectively. A linear correlation was observed between combined thickness of nerve fiber layer (NFL), GCL, and inner plexiform layer (IPL) imaged by SD-OCT and cell numbers in GCL. Only half of the normal BALB/cJ mice exhibited pattern ERG response, which was smaller and later compared to C57BL/6J mice. The ONC reduced pattern ERG and pSTR, but the reduction of pattern ERG was earlier than pSTR, preceding the anatomical cell loss in the GCL. The PhNR and scotopic OPs were not affected by ONC., Conclusions: The SD-OCT and ERG can be used to monitor noninvasively retinal morphologic and functional changes induced by ONC. Pattern ERG and pSTR are able to detect early RGC dysfunction, but pattern ERG exhibits higher sensitivity. Our results support the use of these tools in studies using the mouse ONC model., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

The aim of this study was to evaluate the degeneration and regeneration of a sensory nerve and a motor nerve at the histological level after a crush injury. Twenty-five female Wistar rats had their mental nerve and the buccal branch of their facial nerve compressed unilaterally against a glass rod for 30s. Specimens of the compressed nerves and the corresponding control nerves were dissected at 3, 7, and 19 days after surgery. Nerve cross-sections were stained with osmium tetroxide and toluidine blue and analysed using two-dimensional stereology. We found differences between the two nerves both in the normal anatomy and in the regenerative pattern. The mental nerve had a larger cross-sectional area including all tissue components. The mental nerve had a larger volume fraction of myelinated axons and a correspondingly smaller volume fraction of endoneurium. No differences were observed in the degenerative pattern; however, at day 19 the buccal branch had regenerated to the normal number of axons, whereas the mental nerve had only regained 50% of the normal number of axons. We conclude that the regenerative process is faster and/or more complete in the facial nerve (motor function) than it is in the mental nerve (somatosensory function)., (Copyright © 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Mature retinal ganglion cells (RGCs) do not normally regenerate injured axons, but undergo apoptosis soon after axotomy. Besides the insufficient intrinsic capability of mature neurons to regrow axons inhibitory molecules located in myelin of the central nervous system as well as the glial scar forming at the site of injury strongly limit axon regeneration. Nevertheless, RGCs can be transformed into a regenerative state upon inflammatory stimulation (IS), enabling these neurons to grow axons into the injured optic nerve. The outcome of IS stimulated regeneration is, however, still limited by the inhibitory extracellular environment. Here, we report that the chemokine CXCL12/SDF-1 moderately stimulates neurite growth of mature RGCs on laminin in culture and, in contrast to CNTF, exerts potent disinhibitory effects towards myelin. Consistently, co-treatment of RGCs with CXCL12 facilitated CNTF stimulated neurite growth of RGCs on myelin. Mature RGCs express CXCR4, the cognate CXCL12 receptor. Furthermore, the neurite growth promoting and disinhibitory effects of CXCL12 were abrogated by a specific CXCR4 antagonist and by inhibition of the PI3K/AKT/mTOR-, but not the JAK/STAT3-pathway. In vivo, intravitreal application of CXCL12 sustained mTOR activity in RGCs upon optic nerve injury and moderately stimulated axon regeneration in the optic nerve without affecting the survival of RGCs. Importantly, intravitreal application of CXCL12 also significantly increased IS triggered axon regeneration in vivo. These data suggest that the disinhibitory effect of CXCL12 towards myelin may be a useful feature to facilitate optic nerve regeneration, particularly in combination with other axon growth stimulatory treatments., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In adult mammals, after optic nerve injury, retinal ganglion cells (RGCs) do not regenerate their axons and most of them die by apoptosis within a few days. Recently, several strategies that activate neuronal intracellular pathways were proposed to prevent such degenerative processes. The rho-related small GTPase Rac1 is part of a complex, still not fully understood, intracellular signaling network, mediating in neurons many effects, including axon growth and cell survival. However, its role in neuronal survival and regeneration in vivo has not yet been properly investigated. To address this point we intravitreally injected selective cell-penetrating Rac1 mutants after optic nerve crush and studied the effect on RGC survival and axonal regeneration. We injected two well-characterized L61 constitutively active Tat-Rac1 fusion protein mutants, in which a second F37A or Y40C mutation confers selectivity in downstream signaling pathways. Results showed that, 15 days after crush, both mutants were able to improve survival and to prevent dendrite degeneration, while the one harboring the F37A mutation also improved axonal regeneration. The treatment with F37A mutant for one month did not improve the axonal elongation respect to 15 days. Furthermore, we found an increase of Pak1 T212 phosphorylation and ERK1/2 expression in RGCs after F37A treatment, whereas ERK1/2 was more activated in glial cells after Y40C administration. Our data suggest that the selective activation of distinct Rac1-dependent pathways could represent a therapeutic strategy to counteract neuronal degenerative processes in the retina.

Introduction: The cavernous nerve (CN) is commonly injured during prostatectomy, resulting in erectile dysfunction (ED). Although peripheral nerves have a limited ability to regenerate, a return of function typically does not occur due to irreversible down stream morphological changes in the penis that result from CN injury. We have shown in previous studies that sonic hedgehog (SHH) is critical for CN regeneration and improves erectile function after crush injury., Aims: Examine a new direction, to determine if SHH is neuroprotective to the pelvic ganglia (PG)/CN after crush injury. A secondary focus is to examine if SHH signaling decreases with age in the PG/CN., Methods: Sprague-Dawley rats underwent bilateral CN crush and SHH and glial fibrillary acidic protein were quantified by western analysis of the PG/CN (N = 6 rats at each time point) at 1, 2, 4, 7, and 14 days, and the apoptotic index was measured in the penis. SHH was quantified by western in the PG/CN with blockade of anterograde transport (N = 4 rats) in comparison to mouse IgG (N = 4 rats). If SHH is neuroprotective was examined at 4 (N = 14 rats) and 7 days (N = 16 rats) of treatment after CN crush. SHH protein was quantified in aging (P200-300, N = 5 rats) PG/CN in comparison to normal adult (P115-120, N = 3 rats) PG/CN. Main Outcome Measures.  SHH pathway was examined in PG via immunohistochemistry, in situ, western, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)., Results: SHH is neuroprotective in the PG/CN with injury. SHH localization in the PG/CN suggests SHH interaction in neuronal/glial signaling. SHH protein is significantly decreased in the PG/CN after crush injury and in the aged PG/CN. Signals from the PG are required to maintain SHH in the CN., Conclusions: There is a window of opportunity immediately after nerve insult in which manipulation of SHH signaling in the nerve microenvironment can affect long-term regeneration outcome., (© 2012 International Society for Sexual Medicine.)

Purpose: Innate immunity plays a role in many diseases, including glaucoma and AMD. We have used transcriptome profiling in the mouse to identify a network of genes involved in innate immunity that is present in the normal retina and that is activated by optic nerve crush (ONC)., Methods: Using a recombinant inbred (RI) mouse strain set (BXD, C57BL/6 crossed with DBA/2J mice), we generate expression datasets (Illumina WG 6.2 arrays) in the normal mouse retina and 2 days after ONC. The normal dataset is constructed from retinas from 80 mouse strains and the ONC dataset is constructed from 62 strains. These large datasets are hosted by GeneNetwork.org, along with a series of powerful bioinformatic tools., Results: In the retina datasets, one intriguing network involves transcripts associated with the innate immunity. Using C4b to interrogate the normal dataset, we can identify a group of genes that are coregulated across the BXD RI strains. Many of the genes in this network are associated with the innate immune system, including Serping1, Casp1, C3, Icam1, Tgfbr2, Cfi, Clu, C1qg, Aif1, and Cd74. Following ONC, the expression of these genes is upregulated, along with an increase in coordinated expression across the BXD strains. Many of the genes in this network are risk factors for AMD, including C3, EFEMP1, MCDR2, CFB, TLR4, HTA1, and C1QTNF5., Conclusions: We found a retina-intrinsic innate immunity network that is activated by injury including ONC. Many of the genes in this network are risk factors for retinal disease.

Peripheral neuropathy and insensate limbs and digits cause significant morbidity in diabetic individuals. Previous studies showed that deletion of the receptor for advanced end-glycation products (RAGE) in mice was protective in long-term diabetic neuropathy. Here, we tested the hypothesis that RAGE suppresses effective axonal regeneration in superimposed acute peripheral nerve injury attributable to tissue-damaging inflammatory responses. We report that deletion of RAGE, particularly in diabetic mice, resulted in significantly higher myelinated fiber densities and conduction velocities consequent to acute sciatic nerve crush compared with wild-type control animals. Consistent with key roles for RAGE-dependent inflammation, reconstitution of diabetic wild-type mice with RAGE-null versus wild-type bone marrow resulted in significantly improved axonal regeneration and restoration of function. Diabetic RAGE-null mice displayed higher numbers of invading macrophages in the nerve segments postcrush compared with wild-type animals, and these macrophages in diabetic RAGE-null mice displayed greater M2 polarization. In vitro, treatment of wild-type bone marrow-derived macrophages with advanced glycation end products (AGEs), which accumulate in diabetic nerve tissue, increased M1 and decreased M2 gene expression in a RAGE-dependent manner. Blockade of RAGE may be beneficial in the acute complications of diabetic neuropathy, at least in part, via upregulation of regeneration signals.

Purpose: The sigma-1 receptor (σR1), a ligand-operated chaperone, has been inferred to be neuroprotective in previous studies using σR1 ligands. The σR1 specificity of the protective function, however, has yet to be firmly established, due to the existence of non-σR1 targets of the ligands. Here, we used the σR1-knockout mouse (Sigmar1(-/-)) to demonstrate unambiguously the role of the σR1 in protecting the retinal ganglion cells against degeneration after acute damage to the optic nerve., Methods: Retinal σR binding sites were labeled with radioiodinated σR ligands and analyzed by autoradiography. Localization of the σR1 was performed by indirect immunofluorescence on frozen retinal sections. Retinal ganglion cell death was induced by acute optic nerve crush in wild-type and Sigmar1(-/-) mice. Surviving cells in the ganglion cell layer were counted on Nissl-stained retinal whole mounts 7 days after the crush surgery., Results: Photoaffinity labeling indicated the presence of the σR1 in the retina, in concentrations equivalent to those in liver tissue. Immunolabeling detected this receptor in cells of both the ganglion cell layer and the photoreceptor cell layer in wild-type retinas. Quantification of cells remaining after optic nerve crush showed that 86.8±7.9% cells remained in the wild-type ganglion cell layer, but only 68.3±3.4% survived in the Sigmar1(-/-), demonstrating a significant difference between the wild-type and the Sigmar1(-/-) in crush-induced ganglion cell loss., Conclusions: Our data indicated faster retinal ganglion cell death in Sigmar1(-/-) than in wild-type mice under the stresses caused by optic nerve crush, providing direct evidence for a role of the σR1 in alleviating retinal degeneration. This conclusion is consistent with the previous pharmacological studies using σR1 agonists. Thus, our study supports the idea that the σR1 is a promising therapeutic target for neurodegenerative retinal diseases, such as glaucoma.

Object: Extradural and intraneural scar formation after peripheral nerve injury frequently causes tethering and compression of the nerve as well as inhibition of axonal regeneration. Regenerating agents (RGTAs) mimic stabilizing and protective properties of sulphated glycosaminoglycan toward heparin-binding growth factors. The aim of this study was to assess the effect of an RGTA known as OTR4120 on extraneural fibrosis and axonal regeneration after crush injury in a rat sciatic nerve model., Methods: Thirty-two female Wistar rats underwent a standardized crush injury of the sciatic nerve. The animals were randomly allocated to RGTA treatment or sham treatment in a blinded design. To score neural adhesions, the force required to break the adhesions between the nerve and its surrounding tissue was measured 6 weeks after nerve crush injury. To assess axonal regeneration, magnetoneurographic measurements were performed after 5 weeks. Static footprint analysis was performed preoperatively and at Days 1, 7, 14, 17, 21, 24, 28, 35, and 42 postoperatively., Results: The magnetoneurographic data show no significant difference in conduction capacity between the RGTA and the control group. In addition, results of the static footprint analysis demonstrate no improved or accelerated recovery pattern. However, the mean pullout force of the RGTA group (67 +/- 9 g [mean +/- standard error of the mean]) was significantly (p < 0.001) lower than that of the control group (207 +/- 14 g [mean +/- standard error of the mean])., Conclusions: The RGTAs strongly reduce nerve adherence to surrounding tissue after nerve crush injury.

Objective: To investigate the relation between the quantitative assessment of magnetic resonance imaging (MRI) features and the correlation with histology and functional recovery by using the rabbit sciatic nerve crush model., Methods: In New Zealand, 32 rabbits were randomly divided into 2 groups (group A and B); all rabbits underwent crushing injury of their left sciatic nerve. In group A (n = 16), the sciatic nerves were crushed by using microvessel clamps with a strength of 3.61 kg. In group B (n = 16), the sciatic nerves were crushed with a strength of 10.50 kg. Right sciatic nerves were served as controls. Serial MRI of both hind limbs in each rabbit was performed before and at the time point of 1, 2, 4, and 8 weeks after crushed injury. The MRI protocol included T1-weighted spin-echo (T1WI), 3 dimension turbo spin-echo T2-weighted (3DT2WI), T2-weighted turbo spin-echo images with spectral presaturation with inversion recovery (T2WI/SPIR), balanced fast-field echo (B-FFE) and short-time inversion recovery (STIR) sequences. The coronal image of the sciatic nerve was obtained. The nerve and muscle signal ratio (SIR) on each sequence was measured. The function recovery was observed and pathological examination was performed at each time point., Results: A signal intensity increase of the distal segment of crushed sciatic nerves was found on 3DT2WI, T2WI/SPIR, B-FFE, and STIR, but not on T1WI images. Of 32 crushed nerves, 30 nerves showed high signal intensity. The correct diagnostic rate was 93.75% with false negative-positive of 6.25%. The SIR of the crushed sciatic nerve at distal portion was higher than those of the control nerves; there was a statistically significant difference (P < 0.001). The SIR of the distal portion of crushed nerves was higher than that of the proximal nerve portion; there was a statistically significant difference (P < 0.001). Whereas, the SIR at proximal nerve portions of crushed nerve was similar to control nerves (P > 0.05). The SIR between group A and group B was not found statistically significantly different (P > 0.05). The SIR of crushed nerves at distal portion increased at one week after the crush injury, subsequently further increased, and reached a maximum at 2 weeks. The pathological examination revealed myelin swelling and axonal fragmentation of crushed nerve. Abduction function of injured hind limb was deficit. From 4 to 8 weeks following the crush, the SIR decreased, correspondingly, nerve regeneration was revealed on pathology including extensive Schwann cells proliferation and the immature myelin formation. The abduction function gradually recovered. There was no abnormal finding on MRI for control and sham-operated nerves., Conclusion: The SIR of injured nerve at distal portion increased on MRI. The evolution of SIR after injury was correlated with the degeneration and regeneration of nerve and the function recovery of lower extremities. Assessment of peripheral nerve injury by using SIR could reveal acute nerve injury, as well as aid in monitoring the recovery process. The pathophysiological basis for the SIR is predominantly the results of axon breakdown and myelin regeneration

Subthreshold membrane potential oscillations (SMPO) in the injured dorsal root ganglion (DRG) neurons are involved in the generation of spontaneous activity, which can directly evoke neuropathic pain. Nerve injury usually triggers the synthesis of large quantities of membrane protein in nerve injured DRG neurons. Membrane proteins are glycosylated by addition of sugars, especially negatively charged sialic acid residues, which may depolarize the resting membrane potential (Vm), open voltage-gated channels in injured neurons, and cause spontaneous activity. In the present study, we aimed to determine if increased negative charge on the cell surface, carried by the sialic acid residues, could contribute to the generation of SMPO in injured DRG neurons. Intracellular recording was performed in DRG neurons following chronic constrictive injury (CCI) of the sciatic nerve. Results indicated that both A- and C-type injured DRG neurons exhibited a higher incidence of SMPO and more depolarized Vm than those of the control neurons. Ca(2+), Mg(2+), Mn(2+), or poly-lysine, a positively charged organic compound, when topically applied to the DRG, not only reduced SMPO but also caused a rapid hyperpolarizing shift in Vm. Topical application of neuraminidase to selectively remove sialic acid residues on the extracellular membrane normalized the depolarized Vm and inhibited both spontaneous and evoked SMPO. However, application of Ca(2+), Mg(2+), Mn(2+) or neuraminidase had no effect on excitability and Vm in normal neurons. The results demonstrated that the increase in negatively charged sialic acid residues on the extracellular membrane of neuronal somata is a critical factor in the generation of SMPO and hyperexcitability in injured sensory neurons.

Using intracellular recording in vivo, the bursting behaviors were investigated in the neurons of chronically compressed dorsal root ganglia of the adult rat. In most cases, the first spike of a burst emerged from amplitude-increasing damped subthreshold membrane potential oscillation (SMPO) and the discharge terminated by an amplitude-decreasing damped SMPO. The rhythms of these bursting behaviors are all irregular. Since some researchers found that the stochastic dynamics can also produce similar bursting pattern, the deterministic dynamics of interevent interval (IEI) series obtained from raw membrane potential recording was detected by extraction of the hierarchy of unstable periodic orbits (UPOs) in the windowed IEI series. The results showed a number of statistically significant UPOs of order-one, order-two, and order-three. These orbits form a complex but predictable lattice of regions in which the dynamics of the bursting occurrence is deterministic. Based on a complete classification scheme, the investigated bursting can be depicted by the elliptic bursting dynamics. The significance of the finding that a neuron in the injured dorsal root ganglion has such dynamics is also discussed., (Copyright 2004 S. Karger AG, Basel)

Object: In a number of clinical studies magnetic resonance (MR) neurography has been used to examine patients with peripheral nerve damage, but little is understood about the sequence of imaging changes following nerve injuries, and how they correlate with functional deficit. The goal of this study was to further understanding of these changes and their implications., Methods: Using the rat sciatic nerve crush model, the sciatic nerve was imaged at intervals over 70 days in 12 rats. Sham-operated contralateral nerves served as controls. A 4.7-tesla MR imager with a custom-made surface coil was used. The T2 maps were calculated from images obtained at four echo times and from regions of interest designated on the nerve at three sites. Walking-track analysis was performed at the same intervals as imaging. Magnetic resonance neurography revealed a mean T2 of normal sciatic nerve of 36 msec (standard deviation [SD] 1.2 msec). Crushed nerves demonstrated a sequence of changes in signal intensity that were maximal at 14 days, with a mean T2 of 64 msec (SD 5.2 msec), then falling to a T2 of 53 msec (SD 3.7 msec). Sham-operated nerves had a short and nonsustained rise in signal at 7 days. Walking-track analysis revealed maximum deficit immediately postinjury, with an improvement in function approaching that of control nerves at 30 days., Conclusions: In this study the authors demonstrate that quantitative assessment of nerve signals with MR neurography allows the sequence of events following nerve crush injury to be followed in vivo, and that a return toward a normal signal correlates with functional improvement. Assessment of peripheral nerve injury in patients by using MR neurography has the potential to confirm acute nerve injury as well as to monitor the recovery process.

Extracellular levels of adenosine (ADO) can be raised through inhibition of adenosine kinase (AK), a primary metabolic enzyme for ADO. AK inhibitors have shown antinociceptive activity in a variety of animal models of nociception. The present study investigated the antinociceptive actions of a novel and selective AK inhibitor, A-134974 (IC(50)=60 pM), in a rat model of neuropathic pain (ligations of the L5/L6 spinal nerves) and explored the relative contributions of supraspinal, spinal and peripheral sites to the actions of A-134974. Systemic A-134974 dose-dependently reduced tactile allodynia (ED(50)=5 micromol/kg, i.p.) for up to 2 h. Fall latencies in the rotorod test of motor coordination were unaffected by systemic administration of A-134974 (at doses up to 30 micromol/kg, i.p.). Administration of A-134974 intrathecally (i.t.) was more potent (ED(50)=10 nmol) in relieving tactile allodynia than delivering the compound by intracerebroventricular (ED(50)>100 nmol, i.c.v.) or intraplantar (ED(50)>500 nmol) routes suggesting that spinal sites of action are the primary contributors to the anti-allodynic action of A-134974. The anti-allodynic effects of systemic A-134974 (10 micromol/kg, i.p.) were antagonized by the non-selective ADO receptor antagonist, theophylline (30-500 nmol) administered i.t. These data demonstrate that the novel AK inhibitor A-134974 potently reduces tactile allodynia through interactions with spinal sites and adds to the growing evidence that AK inhibitors may be useful as analgesic agents in a broad spectrum of pain states.

Changes in phenotype or connectivity of primary afferent neurons following peripheral nerve injury may contribute to the hyperalgesia and allodynia associated with neuropathic pain conditions. Although earlier studies using partial nerve injury models have focused on the role of damaged fibres in the generation of ectopic discharges and pain, it is now thought that remaining undamaged fibres may be equally important. We have examined the expression of the sensory neuron-specific cation channel Vanilloid Receptor 1 (VR1), an important transducer of noxious stimuli, in three models of nerve injury in the rat, using anatomical separation or fluorescent retrograde tracers to identify damaged or undamaged sensory neurons. After total or partial sciatic nerve transection, or spinal nerve ligation, VR1-immunoreactivity (IR) was significantly reduced in the somata of all damaged dorsal root ganglion (DRG) neuronal profiles, compared to controls. However, after partial transection or spinal nerve ligation, VR1 expression was greater in the undamaged DRG somata than in controls. Unexpectedly, after L5 spinal nerve ligation, VR1-IR of the A-fibre somata increased approximately 3-fold in the uninjured L4 DRG compared to controls; a much greater increase than seen in the somata with C-fibres. Furthermore, we found that VR1-IR persisted in the transected sciatic nerve proximal to the lesion, despite its down-regulation in the damaged neuronal somata. This persistence in the nerve proximal to the lesion after nerve section, together with increased VR1 in DRG neurons left undamaged after partial nerve injury, may be crucial to the development or maintenance of neuropathic pain.

An abundance of subthreshold membrane potential oscillations (SMPOs) at resting potential was observed in the neurons of chronically compressed dorsal root ganglia (DRG) using intracellular recording in vivo. Out of 386 neurons, 63 type A neurons displayed SMPOs. Three types of SMPOs were distinguished based on their characterizations of oscillation: (1) A regular pattern of SMPO emerged consistently with a mean frequency of 86 Hz and mean amplitudes of 3.3 mV. (2) A spindle-like pattern of SMPO was denominated by a spindle alteration of its amplitude. (3) An irregular pattern of SMPO had no rule on its change of amplitude and frequency. Compared with normal DRG neurons and injured DRG neurons but without SMPO, the injured DRG neurons with SMPO had the lowest spike rheobase, in accordance with the detection of spike accommodation. No significant differences among the three groups can be found in either membrane potential or input resistance. Further observation showed that the spontaneous discharge of hyperexcitable neurons usually occurred on the depolarizing phase of oscillations. In addition, the regular pattern of SMPO was based on the period and integer multiple patterns of spontaneous discharges. The spindle-like pattern of SMPO contributed to spontaneous bursting discharge. The irregular pattern of SMPO had a striking relation with irregular spontaneous discharge. The results show that neurons with SMPO in injured DRG have a higher excitability than those without SMPO, and that the SMPO underlie the patterns of spontaneous discharges, suggesting that SMPO is the basic electrophysiological change of hyperexcitable neurons.

It is well documented that there is an increase in the number of sympathetic fibers within the dorsal root ganglion (DRG) after a peripheral nerve injury. The present study examined the numbers and distribution of sympathetic fibers in the DRG and their sprouting routes by utilizing various surgical manipulations and retrograde tracing and immunohistochemical staining methods in spinal nerve-ligated neuropathic rats. The appearance of many double immunostained fibers with antibodies to tyrosine hydroxylase (TH) and growth associated protein-43 (GAP-43) in the L5 DRG 1 week after L5 spinal nerve ligation, indicated sprouting of sympathetic fibers. The confined location of early sprouting sympathetic fibers in the distal half of the L5 DRG confirmed that sprouting fibers come primarily from the injured spinal nerve. A second cut proximal to the previously ligated L5 spinal nerve -- a process which would transect the regenerating sympathetic fibers extending from the injury site -- did not change the density of sympathetic fibers in the L5 DRG. When retrograde tracers (fast blue and diamidino yellow) were injected into the L5 spinal nerve and DRG, respectively, the number of double-labeled sympathetic postganglionic neurons was greatly increased after spinal nerve ligation, suggesting the increased number of sympathetic neurons projecting to both the spinal nerve and DRG. All these results indicate that many sympathetic fibers in the DRG are regenerating branches that are sprouting from the proximal part of the injured spinal nerve (regenerative collateral sprouting).

This study was aimed to investigate the possible involvement of neurons in the cuneate nucleus (CN) in the processing of A beta afferent inputs evoked by electrical stimulation of constricted median nerve in rats with behavioral signs of neuropathic pain. Immunohistochemical localization of Fos protein was used to examine the neuronal activation, and the combination of Fos immunohistochemistry with the retrograde labeling of Fluoro-Gold (FG) injected into the ventrobasal complex of the thalamus was used to characterize the activated neurons. Two weeks after unilateral median nerve constriction injury, the rats exhibited behavioral signs of neuropathic pain in the affected forepaws. In rats after nerve injury but without electrical stimulation, some Fos-like immunoreactive (Fos-LI) neurons were detected in the dorsal horn of the seventh cervical segment (C7) but none was found in the CN. Similar features were also noted when the stimulation of the intact median nerve served as an additional control. After A beta-fiber intensity stimulation of the previously constricted median nerve, an increase in number of Fos-LI neurons occurred in the medial half of the ipsilateral C7 dorsal horn as well as in the ipsilateral CN. In the latter, the Fos-LI neurons were located in the median nerve projection territory throughout the nucleus. Most of the Fos-LI neurons were distributed in the middle region of the CN, with about 78% of them emitting FG fluorescence indicating that they were cuneothalamic projection neurons. The results of this study suggest that the dorsal column-medial lemniscal system may contribute to the transmission and modulation of A beta-fiber mediated neuropathic pain signals.

In peripheral nerves, progesterone synthesized by Schwann cells has been implicated in myelination. In spite of such an important function, little is known of the regulation of progesterone biosynthesis in the nervous system. We show here that in rat Schwann cells, expression of the 3 beta-hydroxysteroid dehydrogenase and formation of progesterone are dependent on neuronal signal. Levels of 3 beta-hydroxysteroid dehydrogenase mRNA and synthesis of [3H]progesterone from [3H]pregnenolone were low in purified Schwann cells prepared from neonatal rat sciatic nerves. However, when Schwann cells were cultured in contact with sensory neurons, both expression and activity of the 3 beta-hydroxysteroid dehydrogenase were induced. Regulation of 3 beta-hydroxysteroid dehydrogenase expression by neurons was also demonstrated in vivo in the rat sciatic nerve. 3 beta-hydroxysteroid dehydrogenase mRNA was present in the intact nerve, but could no longer be detected 3 or 6 days after cryolesion, when axons had degenerated. After 15 days, when Schwann cells made new contact with the regenerating axons, the enzyme was re-expressed. After nerve transection, which does not allow axonal regeneration, 3 beta-hydroxysteroid dehydrogenase mRNA remained undetectable. The regulation of 3 beta-hydroxysteroid dehydrogenase mRNA after lesion was similar to the regulation of myelin protein zero (P0) and peripheral myelin protein 22 (PMP22) mRNAs, supporting an important role of locally formed progesterone in myelination.

We investigated whether lowering barometric pressure by 20 mmHg (LP) aggravates the guarding behavior suggestive of spontaneous pain following sciatic nerve chronic constriction injury (CCI) in rats. Systemic blood pressure (BP) and heart rate (HR) of unrestrained rats were recorded telemetrically during LP both before and after the CCI surgery. CCI rats showed guarding posture in normopressure conditions, and LP increased the cumulative time of this behavior. Baseline BP but not HR was increased following CCI. LP increased BP and HR of the rats only before the CCI surgery. Animals after CCI surgery showed variable (BP, HR) and transient (HR) responses to LP. These results indicate that (1) LP aggravated spontaneous pain and increased BP and HR in the CCI rats, and (2) CCI surgery influenced BP and HR of rats.

Small conductance, calcium-activated potassium channels (SK channels) are present in most neurons, in denervated muscles and in several non-excitable cell types. In excitable cells SK channels play a fundamental role in the generation of the afterhyperpolarization which follows an action potential, thereby modulating neuronal firing and regulating excitability. To date, three channel subunits (SK1-3) have been cloned from mammalian brain. Since SK3 only has been shown to be expressed in muscles upon denervation, this channel may be involved in hyperexcitability and afterhyperpolarization observed in muscle cells in the absence of the nerve. Using confocal microscopy and SK3 specific antibodies, we demonstrate that SK3 immunoreactivity is present at the rat neuromuscular junction in denervated but also in innervated muscles. In denervated muscle fibers, SK3 is localized in the extrajunctional as well as the junctional plasma membrane, where it appears to be less abundant in the acetylcholine receptor-rich domains, corresponding to the crests of the postsynaptic folds. In innervated muscles, SK3 is not detectable in the muscle fiber but is present at the neuromuscular junction and seems to be localized presynaptically in the motor nerve terminals. Axonal accumulation of SK3 immunoreactivity occurs above and below a ligature of rat sciatic nerve, indicating that the SK3 protein is transported in both directions along the axons of the motor neurons. During rat development SK3 immunoreactivity is not found at the neuromuscular junction until day 35 of postnatal development when SK3 first appears in the motor neuron terminals. These results indicate that SK3 channels are components of the presynaptic compartment in the mature neuromuscular junction, where they may play an important regulatory role in synaptic transmission.

Neurons in the superficial laminae of the dorsal horn are dominated by input from peripheral nociceptors. Following peripheral nerve injury, low threshold mechanoreceptive Abeta-fibers sprout from their normal termination site in laminae III/IV into laminae I-II and this structural reorganization may contribute to neuropathic tactile pain hypersensitivity. We have now investigated whether a sciatic nerve crush injury alters the behavioral response in rats to tactile stimuli and whether this is associated with a change in the pattern of c-Fos expression in the dorsal horn and the parabrachial area of the brainstem. Sciatic nerve crush resulted in a patchy but marked tactile allodynia manifesting first at 3 weeks and persisting for up to 52 weeks. C-Fos expression in the dorsal horn and parabrachial region was never observed on brushing the skin of the sciatic nerve territory in animals with intact nerves, but was found after sciatic nerve crush with peripheral regeneration. We conclude that after nerve injury, low threshold mechanoreceptor fibers may play a major role in producing pain-related behavior by activating normally nociceptive-specific regions of the central nervous system such as the superficial laminae of the dorsal horn and the parabrachial area., (Copyright 2000 Wiley-Liss, Inc.)

Injury to a peripheral nerve initiates changes that can lead to regeneration of the damaged axons. How information about a distant injury is communicated to the cell body is not clear. Using the nervous system of Aplysia californica, we tested the idea that some of this information is conveyed via positive injury signals-axoplasmic proteins that are activated at the injury site and transported to the cell soma. We collected these proteins by crushing pedal nerves and then placing a ligation proximal to the ligation. The contralateral nerves were ligated as controls. Twenty h later, axoplasm was extruded from the nerve segment just distal to the ligation on the crushed nerves (cr/lig) and on the control nerves (lig). The total proteins were rhodaminated and injected into the cytoplasm of neurons in vitro to look for nuclear import. Punctate fluorescence was detected in the nucleus of all seven neurons injected with the cr/lig axoplasm. Only two of five neurons injected with lig axoplasm had any fluorescence. Equal amounts of cr/lig and lig axoplasm were then injected directly into the cell bodies of neurons maintained in vitro. The cells injected with cr/lig axoplasm exhibited renewed growth and significantly longer survival: 25.9 +/- 2.1 days (mean +/- SEM: n = 22) relative to the cells injected with lig axoplasm (15.3 +/- 1.2 days; n = 14) and to those that were not injected (12.2 +/- 1.7 days; n = 24). Fractionation of the cr/lig axoplasm indicated that different factors are responsible for growth and survival., (Copyright 2000 John Wiley & Sons, Inc.)

The dy/dy mouse suffers from a form of muscular dystrophy caused by a substantial reduction in laminin alpha2-chain protein, a major component of both muscle and Schwann cell basal laminae. This article examines the effect of laminin alpha2 deficiency on Schwann cell-axon interactions both in vivo at varying intervals after nerve crush, and in vitro, in cocultures of neurons and Schwann cells. The morphological spectrum of aberrant Schwann cell-axon associations seen in uncrushed dy/dy sciatic nerves was recapitulated during regeneration: myelination of regenerating axons was delayed compared with the process in unaffected mice and the relatively few myelin sheaths which were formed in dy/dy distal nerve stumps were often uncompacted. In vitro, Schwann cells dissociated from adult dy/dy sciatic nerves predictably failed to express detectable laminin alpha2-chain and displayed an unusual multipolar morphology. Branching of neurites, in terms both of numbers of terminal branches and of complexity of branching, from dorsal root ganglia neurons grown on dy/dy Schwann cells, was significantly less extensive than that seen when neurons were cocultured with Schwann cells from unaffected littermates, but this effect was reversed by exogenous laminin-2. Our results lend strong support to the view that laminin-2 is essential for establishing and/or maintaining Schwann cell-axon interactions, in normal and in regenerating nerves., (Copyright 2000 Wiley-Liss, Inc.)

During development, a graded expression of ephrin A2 has been implicated in retinotectal map formation. Here we have examined ephrin A2 expression during optic nerve regeneration in the mature goldfish. In the tecta of normal animals, a gradient of ephrin A2 expression is detected in cell bodies within the stratum fibrosum et griseum superficiale with more immunopositive cells caudally than rostrally. The gradient in the mature animal presumably reflects the plasticity associated with continued retinal and tectal neurogenesis. During optic nerve regeneration, expression throughout the tectum is increased by 1 month as a strong rostrocaudal gradient. The gradient declines to normal by 3 months. The up-regulation of ephrin A2 during optic nerve regeneration is likely to be instrumental in reestablishing the retinotectal map., (Copyright 2000 Academic Press.)

This work was undertaken to study whether retinal ganglion cell (RGC) death, which occurs during postnatal development of the mouse retina could aid in assessing the topological and chronological pattern of microglial cell migration. The study was conducted from postnatal day 0 (P0) to adulthood. The fluorescent dyes Fluorogold (FG) or (4-[4-didecylaminostyryl]-N-methylpyridinium iodide (4Di-10ASP) used in this study, were transported retrogradely to the RGC soma when either dye was injected into the superior colliculus (SC) at P0. Some of these labeled RGCs die due to natural apoptosis during this stage of development and are phagocytosed by microglial cells, which move to the site of RGC death, to become labeled with the same dye. The retinas were examined to quantify the microglial cells from P5 to adulthood. In addition, the reaction of microglia to optic nerve crush was studied in adult animals. Both dyes labeled RGCs in the contralateral retina and a few RGCs in the retina ipsilateral to the injected SC. The density of labeled RGCs decreased by 22% between P5 and P7. During this phase, microglial cells become visible as they ingested the fluorescent detritus of the dying RGCs. Microglial cells were evenly distributed across the entire retinal surface and migrated to the outer plexiform layer. Migrating microglia consecutively altered their morphology from the amoeboid to the ramified form. In terms of intracellular storage of the dyes, resident microglial cells retained the fluorescent dye 4Di-10ASP over a period of 12 months. In contrast, FG was completely transferred from the RGCs and microglial cells to intramural cells (pericytes) of the retinal capillaries after 10 months. This resulted in delineation of the entire intraretinal vascular network. Finally, resident retinal microglial cells were also activated by injury to the adult optic nerve and phagocytosed degenerating neurons. Retinal microglial cells can be monitored with vital fluorescent dyes while they migrate across the retina and establish their intra-retinal network. It is possible to label microglia with lipophilic dyes and they remain labeled for a long time. In addition, intramural pericytes can be labeled by slow release of FG from RGCs and microglial cells. The findings suggest that ingested fluorescent dyes having different properties can be used to study different populations of retinal cells in vivo., (Copyright 2000 Wiley-Liss, Inc.)

Irregular burst firing is spontaneously generated by rat dorsal root ganglion neuron after chronic compression. To investigate the time series of burst firing, we recorded interspike intervals (ISIs) of single-unit firing in vitro and processed the ISIs to obtain interevent intervals (IEIs). Then, two non-linear methods were applied to detect deterministic dynamic behaviors in IEI series. No evidence for the existence of determinism was found with non-linear prediction method. Using unstable periodic orbit identification method, significant period-1 orbits were identified in all 10 data, period-2 orbits in eight, and period-3 orbits in six. The results indicate that there exist significant deterministic behaviors in the time series of irregular burst firing from the injured sensory neuron.

We report the case of a 30-year-old woman affected by hereditary neuropathy with liability to pressure palsies (HNPP), who developed a painless left axillary neuropathy after sleeping on her left side, on a firm orthopaedic mattress, in her eighth month of pregnancy. Electromyography (EMG) showing neurogenic signs in the left anterior and middle deltoid, and normal findings in the left teres minor, posterior deltoid and other proximal upper limb muscles, demonstrated that the lesion was at the level of the axillary anterior branch. A direct compression of this branch against the surgical neck of the humerus seems the most likely pathogenic mechanism. This is the first documented description of an axillary neuropathy in HNPP. Knowledge of its possible occurrence may be important for prevention purposes.

A behavioral test paradigm that measures the aversive quality of stimulus-evoked pain in an animal model of neuropathic pain (L5 ligation) was tested for sensitivity to (1) different forces (476 and 202 mN) and frequencies (once every 15 or 30 s) of mechanical stimulation to the hyperalgesic paw and (2) different doses of the common antinociceptive compounds morphine (1 and 10 mg/kg) and gabapentin (30 and 90 mg/kg). Compared to non-ligated controls, the greater force (476 mN) and frequency (every 15 s) of mechanical stimulation of the hyperalgesic paw was associated with the greatest degree of escape/avoidance behavior. There was not a significant degree of escape/avoidance behavior at the lowest force (202 mN) and frequency (every 30 s) of mechanical stimulation. Compared to ligated vehicle treated controls, morphine (1 mg/kg) and gabapentin (90 mg/kg) decreased mechanical hyperalgesia and also attenuated the escape/avoidance behavior. The antinociceptive and antiaversive effects were found at doses that did not produce evidence of decreased motor activity. It is concluded that the behavioral test paradigm used to measure the aversiveness of stimulus-evoked nociceptive behavior is sensitive to different degrees of evoked pain and traditional analgesic compounds.