Immunohistochemical Assessment of GDNF and Chromogranin A Expression in Erosive and Granulomatous Lesions in Glandular Region of Equine Stomach.

The equine stomach consists of two separate non-glandular and glandular sections. Despite the incidence of most lesions in the non-glandular region, both stomach parts are prone to lesions. In this study, 41 hybrid-native horses, including 24 stallions and 17 mares, were examined over five years. In total, 27 horses (65.85%) that were sampled had lesions, including erosion, granuloma, or both on the glandular region of the stomach. Occurrence of gastric erosive and granulomatous lesions had no significant relationship with the age and gender of horses or the sampling season (P>0.05). Moreover, parasites Gastrophilus and Habronema were mainly the primary cause of gastric erosive and granulomatous lesions respectively. In Periodic Acid Schiff (PAS) stained tissue sections, the inflammation severity in granulomatous lesions was higher and statistically significant, compared to erosive lesions (P<0.05). Immunohistochemistry revealed negative expression of glial cell line-derived neurotrophic factor in gastric lesions, while its expression was relatively positive in normal stomachs. Interestingly, based on counting cells and evaluation of expression intensity, Chromogranin A expression in neuroendocrine glandular cells had a significant relationship with the increase of severity and depth of the lesions (P<0.05). The results indicated that the glial cell line-derived neurotrophic factor does not affect the pathogenesis of equine gastric lesions while confirming the role of increment of gastric neuroendocrine cells in lesion progress. Furthermore, the increased expression of Ki67 and p53 proteins in granulomatous lesions, compared to other groups, may be associated with the proliferation and control process of the cells in measures regarding the formation and healing of the lesion.

Novel peripheral role of Nurr-1/GDNF/AKT trajectory in carvedilol and/or morin hydrate hepatoprotective effect in a model of hepatic ischemia/reperfusion.

Although the central role of Nurr-1/GDNF has been reviewed amply, scarce data are available on their peripheral impact. Carvedilol and morin hydrate have previously conferred their hepatic anti-fibrotic action. AIM: Thus, our aim was to unveil the potential hepatoprotective role of carvedilol (CR) and/or morin hydrate (MH) using a hepatic 70% partial warm ischemia/reperfusion (I/R) rat model. MAIN METHOD: Rats were allocated into sham-operated, hepatic I/R, and I/R preceded by oral administration of CR (10 and 30 mg/kg; CR10/CR30), MH (30 mg/kg), or CR10 + MH for one week. KEY FINDINGS: On the molecular level, pretreatment with CR and/or MH increased the hepatic contents of Nurr-1, GDNF, and the protein expression of active/p-AKT. On the other hand, they inactivated GSK3ß and NF-κB to increase the antioxidant enzymes (GPx, SOD, CAT). All regimens also enhanced the autophagy/lysosomal function and boosted the protein expression of beclin-1, LC3II, and TFEB. Moreover, their antiapoptotic effect was signified by increasing the anti-apoptotic molecule Bcl2 and inhibiting Bax, Bax/Bcl2 ratio, and caspase-3, effects that were confirmed by the TUNEL assay. These improvements were reflected on liver function, as they decreased serum aminotransferases and liver structural alterations induced by I/R. Despite its mild impact, CR10 showed marked improvements when combined with MH; this synergistic interaction overrides the effect of either regimen alone. SIGNIFICANCE: In conclusion, CR, MH, and especially the combination regimen, conferred hepatoprotection against I/R via activating the Nurr-1/GDNF/AKT trajectory to induce autophagy/lysosomal biogenesis, inhibit GSK3ß/NF-кB hub and apoptosis, and amend redox balance.

Safety Assessment of AAV2-hGDNF Administered Via Intracerebral Injection in Rats for Treatment of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neuroprotective biologic in Parkinson's disease models. Adeno-associated viral vector serotype 2 (AAV2)-human GDNF safety was assessed in rats treated with a single intracerebral dose of vehicle, 6.8 × 108, 6.8 × 109, or 5.2 × 1010 vector genomes (vg)/dose followed by interim sacrifices on day 7, 31, 90, and 376. There were no treatment-related effects observed on food consumption, body weight, hematology, clinical chemistry, coagulation parameters, neurobehavioral parameters, organ weights, or serum GDNF and anti-GDNF antibody levels. Increased serum anti-AAV2 neutralizing antibody titers were observed in the 5.2 × 1010 vg/dose group. Histopathological lesions were observed at the injection site in the 6.8 × 109 vg/dose (day 7) and 5.2 × 1010 vg/dose groups (days 7 and 31) and consisted of gliosis, mononuclear perivascular cuffing, intranuclear inclusion bodies, and/or apoptosis on day 7 and mononuclear perivascular cuffing on day 31. GDNF immunostaining was observed in the injection site in all dose groups through day 376 indicating no detectable impacts of anti-AAV2 neutralizing antibody. There was no evidence of increased expression of calcitonin gene-related peptide or Swann cell hyperplasia in the cervical and lumbar spinal cord or medulla oblongata at the 5.2 × 1010 vg/dose level indicating lack of hyperplastic effects. In conclusion, no systemic toxicity was observed, and the local toxicity observed at the injection site appeared to be reversible demonstrating a promising safety profile of intracerebral AAV2-GDNF delivery. Furthermore, an intracerebral dose of 6.8 × 108 AAV2-GDNF vg/dose was considered to be a no observed adverse effect level in rats.

Assessment of Glial Scar, Tissue Sparing, Behavioral Recovery and Axonal Regeneration following Acute Transplantation of Genetically Modified Human Umbilical Cord Blood Cells in a Rat Model of Spinal Cord Contusion.

OBJECTIVE AND METHODS: This study investigated the potential for protective effects of human umbilical cord blood mononuclear cells (UCB-MCs) genetically modified with the VEGF and GNDF genes on contusion spinal cord injury (SCI) in rats. An adenoviral vector was constructed for targeted delivery of VEGF and GDNF to UCB-MCs. Using a rat contusion SCI model we examined the efficacy of the construct on tissue sparing, glial scar severity, the extent of axonal regeneration, recovery of motor function, and analyzed the expression of the recombinant genes VEGF and GNDF in vitro and in vivo. RESULTS: Transplantation of UCB-MCs transduced with adenoviral vectors expressing VEGF and GDNF at the site of SCI induced tissue sparing, behavioral recovery and axonal regeneration comparing to the other constructs tested. The adenovirus encoding VEGF and GDNF for transduction of UCB-MCs was shown to be an effective and stable vehicle for these cells in vivo following the transplantation into the contused spinal cord. CONCLUSION: Our results show that a gene delivery using UCB-MCs-expressing VEGF and GNDF genes improved both structural and functional parameters after SCI. Further histological and behavioral studies, especially at later time points, in animals with SCI after transplantation of genetically modified UCB-MCs (overexpressing VEGF and GDNF genes) will provide additional insight into therapeutic potential of such cells.

Assessment of TTX-s and TTX-r Action Potential Conduction along Neurites of NGF and GDNF Cultured Porcine DRG Somata.

Nine isoforms of voltage-gated sodium channels (NaV) have been characterized and in excitable tissues they are responsible for the initiation and conduction of action potentials. For primary afferent neurons residing in dorsal root ganglia (DRG), individual neurons may express multiple NaV isoforms extending the neuron's functional capabilities. Since expression of NaV isoforms can be differentially regulated by neurotrophic factors we have examined the functional consequences of exposure to either nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) on action potential conduction in outgrowing cultured porcine neurites of DRG neurons. Calcium signals were recorded using the exogenous intensity based calcium indicator Fluo-8®, AM. In 94 neurons, calcium signals were conducted along neurites in response to electrical stimulation of the soma. At an image acquisition rate of 25 Hz it was possible to discern calcium transients in response to individual electrical stimuli. The peak amplitude of electrically-evoked calcium signals was limited by the ability of the neuron to follow the stimulus frequency. The stimulus frequency required to evoke a half-maximal calcium response was approximately 3 Hz at room temperature. In 13 of 14 (93%) NGF-responsive neurites, TTX-r NaV isoforms alone were sufficient to support propagated signals. In contrast, calcium signals mediated by TTX-r NaVs were evident in only 4 of 11 (36%) neurites from somata cultured in GDNF. This establishes a basis for assessing action potential signaling using calcium imaging techniques in individual cultured neurites and suggests that, in the pig, afferent nociceptor classes relying on the functional properties of TTX-r NaV isoforms, such as cold-nociceptors, most probably derive from NGF-responsive DRG neurons.

Assessment of cone survival in response to CNTF, GDNF, and VEGF165b in a novel ex vivo model of end-stage retinitis pigmentosa.

PURPOSE: To develop a robust ex vivo model for evaluating cone survival in end-stage retinitis pigmentosa (RP) and apply this to quantify the effects of putative neuroprotective compounds. METHODS: Rhodopsin knockout mice were crossed with OPN1-GFP reporter mice so that GFP-positive cones could be identified against the background of a rod-specific degeneration. Retinal explants were harvested from 10-week-old mice and maintained in organotypic culture. Ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), or vascular endothelial growth factor 165b (VEGF(165b)) was administered daily to treatment groups at three doses (200 ng/mL, 100 ng/mL, or 50 ng/mL; n = 5 explants per group). Fluorescence microscopy was performed on days 1, 3, 5, 7, 9, and 12 to document the number of GFP-expressing cones. RESULTS: Cone survival could be assessed reliably and reproducibly in this model, and cone degeneration was significantly greater in the absence of rods, in keeping with clinical observations of RP. Daily administration of 200 ng/mL CNTF led to significantly increased cone survival compared with sham-treated controls. The effect was dose dependent; 100 ng/mL CNTF reduced cone loss but to a lesser extent, and 200 ng/mL GDNF showed significant protection against cone loss at later time points (day 9-12) but was much less effective than CNTF at all doses. VEGF(165b) showed no neuroprotective effect in this model at any dose. CONCLUSIONS: This model allows precise quantification of the neuroprotective effects of various compounds on cone survival and may therefore provide a robust method of screening neuroprotective compounds before application in vivo.

Assessment of GDNF in primate models of Parkinson's disease: comparison with human studies.

Potential future treatments for Parkinson's disease (PD) include those that not only provide symptomatic relief to patients but are also neuroprotective and/or neurorestorative. Glial cell line-derived neurotrophic factor (GDNF) may be a valuable candidate in this regard. Positive results using monkeys have encouraged the use of GDNF in human trials. These trials have unfortunately shown mixed results, illustrating the influence that various parameters of administration can have on clinical outcome. The aim of this review is to compare the findings of these clinical studies with available primate data. While bolus intraventricular injections of GDNF in primates have shown some behavioural efficacy, there was no clinical benefit in the first human trial using this method, which was most likely a result of inefficient GDNF distribution in the striatal parenchyma. In primates, however, continuous (rather than bolus) delivery of GDNF into the ventricles results in significant distribution in the striatum. While chronic delivery of GDNF into the ventricles has not been assessed in humans, intraputamenal protein delivery in two Phase I trials have demonstrated that GDNF considerably reduces PD symptoms, suggesting that the putamen is the optimal location for delivery. Primate studies have shown that vector mediated delivery of GDNF may provide a suitable means for long-term intraputamenal delivery. However, the possibility of high levels of GDNF resulting in widespread distribution of GDNF to non-targeted areas is a cause of concern, and vectors with transgene regulation are needed. The development of these vectors may be the way forward for GDNF treatment.