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.

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.

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.

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.

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.

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.

Treatment with Riluzole Restores Normal Control of Soleus and Extensor Digitorum Longus Muscles during Locomotion in Adult Rats after Sciatic Nerve Crush at Birth.

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.

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.

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.

Behavioral characterization of neuropathic pain on the glabrous skin areas reinnervated solely by axotomy-regenerative axons after adult rat sciatic nerve crush.

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.

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.

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.

Morphometric and high resolution scanning electron microscopy analysis of low-level laser therapy and latex protein (Hevea brasiliensis) administration following a crush injury of the sciatic nerve in rats.

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.

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.

Changes in expression of aquaporin-4 and aquaporin-9 in optic nerve after crushing in rats.

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.

Nerve fiber layer thinning lags retinal ganglion cell density following crush axonopathy.

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.