Regina Rodrigo

p38 MAP kinase is a therapeutic target for hepatic encephalopathy in rats with portacaval shunts

Objective Inflammation plays a role in neurological alterations in patients with hepatic encephalopathy (HE). Animal models of HE show neuroinflammation. Treatment with ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), reduces neuroinflammation and restores cognitive and motor function in rats with HE due to portacaval shunts (PCS). This suggests that reducing neuroinflammation would improve neurological status in patients with minimal or clinical HE. NSAID induce kidney damage in patients with cirrhosis and PCS rats and are not suitable for clinical use. It is therefore necessary to look for procedures to eliminate neuroinflammation without inducing secondary effects in the kidney. Inhibition of p38 MAPK is being tested as a therapeutic target in inflammatory diseases and reduces microglial activation. This study aimed to assess whether inhibiting p38 with SB239063 reduces neuroinflammation and improves cognitive and motor function in PCS rats without affecting the kidney. Results p38 activity is increased in the brains of PCS rats and treatment with SB239063 reduces microglial activation, as well as inflammatory markers in brain (prostaglandin E2, cyclooxygenase activity, iNOS, IL-1β, TNFα) and blood (prostaglandin E2 and TNFα). PCS rats showed increased ammonia and glutamine in the brain, which was not affected by SB239063. PCS rats showed reduced ability to learn a Y-maze conditional discrimination task, reduced motor activity and impaired motor coordination, as assessed in the rotarod. Treatment with SB239063 completely restored learning ability, motor activity and coordination in PCS rats. SB239063 did not affect creatinine or sodium levels in serum, indicating that it does not induce kidney damage. Conclusion These findings suggest that reducing neuroinflammation by using inhibitors of p38 would improve the neurological status in HE without inducing secondary effects in the kidney.

Altered Antioxidant-Oxidant Status in the Aqueous Humor and Peripheral Blood of Patients with Retinitis Pigmentosa

Retinitis Pigmentosa is a common form of hereditary retinal degeneration constituting the largest Mendelian genetic cause of blindness in the developed world. It has been widely suggested that oxidative stress possibly contributes to its pathogenesis. We measured the levels of total antioxidant capacity, free nitrotyrosine, thiobarbituric acid reactive substances (TBARS) formation, extracellular superoxide dismutase (SOD3) activity, protein, metabolites of the nitric oxide/cyclic GMP pathway, heme oxygenase-I and inducible nitric oxide synthase expression in aqueous humor or/and peripheral blood from fifty-six patients with retinitis pigmentosa and sixty subjects without systemic or ocular oxidative stress-related disease. Multivariate analysis of covariance revealed that retinitis pigmentosa alters ocular antioxidant defence machinery and the redox status in blood. Patients with retinitis pigmentosa present low total antioxidant capacity including reduced SOD3 activity and protein concentration in aqueous humor. Patients also show reduced SOD3 activity, increased TBARS formation and upregulation of the nitric oxide/cyclic GMP pathway in peripheral blood. Together these findings confirmed the hypothesis that patients with retinitis pigmentosa present reduced ocular antioxidant status. Moreover, these patients show changes in some oxidative-nitrosative markers in the peripheral blood. Further studies are needed to clarify the relationship between these peripheral markers and retinitis pigmentosa.

Cerebral oedema is not responsible for motor or cognitive deficits in rats with hepatic encephalopathy

Low‐grade cytotoxic oedema is considered a main contributor to the neurological (motor and cognitive) alterations in patients with hepatic encephalopathy (HE). This assumption is mainly based on studies with cultured astrocytes treated with very large ammonia concentrations or with animal models of acute liver failure with strong HE. However, the possible contribution of cerebral oedema (vasogenic or cytotoxic) to cognitive or motor alterations in chronic mild HE has not been demonstrated. The aim of this work was to assess whether cerebral oedema contributes to cognitive and/or motor alterations in rats with chronic mild HE.

Adalimumab reduces photoreceptor cell death in a mouse model of retinal degeneration

Growing evidence suggests that inflammation is involved in the progression of retinitis pigmentosa (RP) both in patients and in animal models. The aim of this study was to investigate the effect of Adalimumab, a monoclonal anti-TNFα antibody, on retinal degeneration in a murine model of human autosomal recessive RP, the rd10 mice at postnatal day (P) 18. In our housing conditions, rd10 retinas were seriously damaged at P18. Adalimumab reduced photoreceptor cell death, as determined by scoring the number of TUNEL-positive cells. In addition, nuclear poly (ADP) ribose (PAR) content, an indirect measure of PAR polymerase (PARP) activity, was also reduced after treatment. The blockade of TNFα ameliorated reactive gliosis, as visualized by decreased GFAP and IBA1 immunolabelling (Müller cell and microglial markers, respectively) and decreased up-regulation of TNFα gene expression. Adalimumab also improved antioxidant response by restoring total antioxidant capacity and superoxide dismutase activity. Finally, we observed that Adalimumab normalized energetic and metabolic pattern in rd10 mouse retinas. Our study suggests that the TNFα blockade could be a successful therapeutic approach to increase photoreceptor survival during the progression of RP. Further studies are needed to characterize its effect along the progression of the disease.

Phosphodiesterase inhibition induces retinal degeneration, oxidative stress and inflammation in cone-enriched cultures of porcine retina

Inherited retinal degenerations affecting both rod and cone photoreceptors constitute one of the causes of incurable blindness in the developed world. Cyclic guanosine monophosphate (cGMP) is crucial in the phototransduction and, mutations in genes related to its metabolism are responsible for different retinal dystrophies. cGMP-degrading phosphodiesterase 6 (PDE6) mutations cause around 4-5% of the retinitis pigmentosa, a rare form of retinal degeneration. The aim of this study was to evaluate whether pharmacological PDE6 inhibition induced retinal degeneration in cone-enriched cultures of porcine retina similar to that found in murine models. PDE6 inhibition was induced in cone-enriched retinal explants from pigs by Zaprinast. PDE6 inhibition induced cGMP accumulation and triggered retinal degeneration, as determined by TUNEL assay. Western blot analysis and immunostaining indicated that degeneration was accompanied by caspase-3, calpain-2 activation and poly (ADP-ribose) accumulation. Oxidative stress markers, total antioxidant capacity, thiobarbituric acid reactive substances (TBARS) and nitric oxide measurements revealed the presence of oxidative damage. Elevated TNF-alpha and IL-6, as determined by enzyme immunoassay, were also found in cone-enriched retinal explants treated with Zaprinast. Our study suggests that this ex vivo model of retinal degeneration in porcine retina could be an alternative model for therapeutic research into the mechanisms of photoreceptor death in cone-related diseases, thus replacing or reducing animal experiments.

Study of USH1 Splicing Variants through Minigenes and Transcript Analysis from Nasal Epithelial Cells

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital profound deafness, vestibular areflexia and prepubertal retinitis pigmentosa. The first purpose of this study was to determine the pathologic nature of eighteen USH1 putative splicing variants found in our series and their effect in the splicing process by minigene assays. These variants were selected according to bioinformatic analysis. The second aim was to analyze the USH1 transcripts, obtained from nasal epithelial cells samples of our patients, in order to corroborate the observed effect of mutations by minigenes in patient’s tissues. The last objective was to evaluate the nasal ciliary beat frequency in patients with USH1 and compare it with control subjects. In silico analysis were performed using four bioinformatic programs: NNSplice, Human Splicing Finder, NetGene2 and Spliceview. Afterward, minigenes based on the pSPL3 vector were used to investigate the implication of selected changes in the mRNA processing. To observe the effect of mutations in the patient’s tissues, RNA was extracted from nasal epithelial cells and RT-PCR analyses were performed. Four MYO7A (c.470G>A, c.1342_1343delAG, c.5856G>A and c.3652G>A), three CDH23 (c.2289+1G>A, c.6049G>A and c.8722+1delG) and one PCDH15 (c.3717+2dupTT) variants were observed to affect the splicing process by minigene assays and/or transcripts analysis obtained from nasal cells. Based on our results, minigenes are a good approach to determine the implication of identified variants in the mRNA processing, and the analysis of RNA obtained from nasal epithelial cells is an alternative method to discriminate neutral Usher variants from those with a pathogenic effect on the splicing process. In addition, we could observe that the nasal ciliated epithelium of USH1 patients shows a lower ciliary beat frequency than control subjects.

Infliximab reduces Zaprinast-induced retinal degeneration in cultures of porcine retina

BackgroundcGMP-degrading phosphodiesterase 6 (PDE6) mutations cause around 4 to 5% of retinitis pigmentosa (RP), a rare form of retinal dystrophy. Growing evidence suggests that inflammation is involved in the progression of RP. The aims of this study were to corroborate the presence of high TNFΑ concentration in the eyes of RP patients and to evaluate whether the blockade of TNFΑ with Infliximab, a monoclonal anti-TNFΑ antibody, prevented retinal degeneration induced by PDE6 inhibition in cultures of porcine retina.MethodsAqueous humor from 30 patients with RP and 13 healthy controls were used to quantify the inflammatory mediators IL-6, TNFΑ, IL-1Β, IL-10 by a multiplex enzyme-linked immunosorbent assay (ELISA) system. Retinal explants from pig were exposed to Zaprinast, a PDE6 inhibitor, for 24 hours in the absence or the presence of Infliximab. Cell death was evaluated by TUNEL assay. The number and distribution of caspase-3 positive cells, indirect poly(ADP)ribose polymerase (PARP) activation and glial fibrillary acidic protein (GFAP) content were visualized by immunolabeling. Antioxidant total capacity, nitrites and thiobarbituric acid reactive substances (TBARS) formation were determined to evaluate antioxidant-oxidant status.ResultsIL-6 and TNFΑ concentrations were higher in the aqueous humor of RP patients than in controls. Infliximab prevented retinal degeneration, as judging by the reduced presence of TUNEL-positive cells, the reduction of caspase-3 activation and also reduction of glial activation, in an ex vivo model of porcine retina. Additionally, Infliximab partially reduced oxidative stress in retinal explants exposed to Zaprinast.ConclusionsInflammatory mediators IL-6 and TNFΑ were elevated in the aqueous humor of RP patients corroborating previous studies suggesting sustained chronic inflammation. Our study suggests that TNFΑ is playing an important role in cell death in an ex vivo model of retinal degeneration by activating different cell pathways at different cell layers of the retina that should be further studied.

cGMP modulates stem cells differentiation to neurons in brain in vivo pathological implications

During brain development there is a strict control of the proliferation, migration and differentiation of neural stem cells to different cell types. Alterations in the control of these processes may result in altered balance in the formation of different cell types resulting in a long-lasting impairment of cerebral processes. This occurs for example if brain is exposed to alcohol during key stages of development which results in accelerated glial cells formation, impaired neuron formation and impaired cognitive function. The molecular mechanisms modulating differentiation of neural stem cells to neurons or non neuronal cells are not well known. Nitric oxide (NO) plays a relevant role in this process. NO increases the activity of soluble guanylate cyclase and is a main modulator of cGMP levels in brain. It has been proposed that cGMP-mediated NO signalling may be involved in the early differentiation events of embryonic stem cells. If this is the case, pathological situations in which the production of cGMP is altered during brain development could lead to altered differentiation of stem cells to neurons or glial cells, resulting in cognitive impairment in the children. Moreover, normalizing cGMP levels in these situations could prevent the alterations in neural stem cells differentiation and cognitive impairment.

HIF-1α stabilization reduces retinal degeneration in a mouse model of retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies characterized by progressive and irreversible loss of vision due to rod and cone degeneration. Evidence suggests that an inappropriate oxygen level could contribute to its pathogenesis. Rod cell death could increase oxygen concentration, reduce hypoxia‐inducible factor 1 (HIF‐1α) and contribute to cone cell death. The purposes of this study were: 1) to analyze the temporal profile of HIF‐1α, its downstream effectors VEGF, endothelin‐1 (ET‐1), iNOS, and glucose transporter 1 (GLUT1), and neuroinflammation in retinas of the murine model of rd10 (retinal degeneration 10) mice with RP; 2) to study oxygen bioavailability in these retinas; and 3) to investigate how stabilizing HIF‐1α proteins with dimethyloxaloglycine (DMOG), a prolyl hydroxylase inhibitor, affects retinal degeneration, neuroinflammation, and antioxidant response in rd10 mice. A generalized down‐regulation of HIF‐1α and its downstream targets was detected in parallel with reactive gliosis, suggesting high oxygen levels during retinal degeneration. At postnatal d 18, DMOG treatment reduced photoreceptor cell death and glial activation. In summary, retinas of rd10 mice seem to be exposed to a hyperoxic environment even at early stages of degeneration. HIF‐1α stabilization could have a temporal neuroprotective effect on photoreceptor cell survival, glial activation, and antioxidant response at early stages of RP.—Olivares‐Gonza´lez, L., Mart´nez‐Fernandezdela Ca´mara, C., Herva´s, D., Milla´n, J. M., Rodrigo, R. HIF‐1α stabilization reduces retinal degeneration in a mouse model of retinitis pigmentosa. FASEB J. 32, 2438–2451 (2018). www.fasebj.org