Combination Therapy with a JNK Inhibitor and Hepatocyte Growth Factor for Restoration of Erectile Function in a Rat Model of Cavernosal Nerve Injury: Comparison with a JNK Inhibitor Alone or Hepatocyte Growth Factor Alone.

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.

Repulsive Environment Attenuation during Adult Mouse Optic Nerve Regeneration.

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.

A Hydroxypyrone-Based Inhibitor of Metalloproteinase-12 Displays Neuroprotective Properties in Both Status Epilepticus and Optic Nerve Crush Animal Models.

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.

Neuroanatomic and behavioral correlates of urinary dysfunction induced by vaginal distension in rats.

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.

α1A-Adrenergic Receptor Antagonism Improves Erectile and Cavernosal Responses in Rats With Cavernous Nerve Injury and Enhances Neurogenic Responses in Human Corpus Cavernosum From Patients With Erectile Dysfunction Secondary to Radical Prostatectomy.

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 α1A-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 α1A-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.

Nerve crush but not displacement-induced stretch of the intra-arachnoidal facial nerve promotes facial palsy after cerebellopontine angle surgery.

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.

Combination histamine and serotonin treatment after simulated childbirth injury improves stress urinary incontinence.

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.

Stimulation of Nitric Oxide Production Contributes to the Antithrombotic Effect of Stromal Cell-Derived Factor-1α in Preventing Microsurgical Anastomotic Thrombosis.

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.

Molecular, anatomical and functional changes in the retinal ganglion cells after optic nerve crush in mice.

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.

CXCL12/SDF-1 facilitates optic nerve regeneration.

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.

Sonic hedgehog is neuroprotective in the cavernous nerve with crush injury.

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.

Accelerated retinal ganglion cell death in mice deficient in the Sigma-1 receptor.

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.

The pudendal nerve motor branch regenerates via a brain derived neurotrophic factor mediated mechanism.

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 ßII 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.

Improved mouse sciatic nerve regeneration following lymphocyte cell therapy.

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.

Significant reduction in neural adhesions after administration of the regenerating agent OTR4120, a synthetic glycosaminoglycan mimetic, after peripheral nerve injury in rats.

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.

Transection and Crush Models of Nerve Injury to Measure Repair and Remyelination in Peripheral Nerve.

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.

Pim-1 Expression in Rat Retina and its Changes after Optic Nerve Crush.

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.

VGF nerve growth factor inducible is involved in retinal ganglion cells death induced by optic nerve crush.

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.

Pelvic and hypogastric nerves are injured in a rat prostatectomy model, contributing to development of stress urinary incontinence.

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.

Glycosylation-induced depolarization facilitates subthreshold membrane oscillation in injured primary sensory neurons.

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.