A hypothermia mimetic molecule (zr17-2) reduces ganglion cell death, gliosis, and electroretinogram distortion in male rats subjected to perinatal asphyxia.

Introduction: Perinatal asphyxia (PA) represents a major problem in perinatology and may cause visual losses, including blindness. We, and others, have shown that hypothermia prevents retinal symptoms associated to PA. In the present work, we evaluate whether a hypothermia mimetic small molecule, zr17-2, has similar effects in the context of PA. Methods: Four experimental groups were studied in male rats: Naturally born rats as controls (CTL), naturally born rats injected s.c. with 50 µL of 330 nmols/L zr17-2 (ZR), animals that were exposed to PA for 20 min at 37°C (PA), and rats that were exposed to PA and injected with zr17-2 (PA-ZR). Forty-five days after treatment, animals were subjected to electroretinography. In addition, morphological techniques (TUNEL, H&E, multiple immunofluorescence) were applied to the retinas. Results: A reduction in the amplitude of the a- and b-wave and oscillatory potentials (OP) of the electroretinogram (ERG) was detected in PA animals. Treatment with zr17-2 resulted in a significant amelioration of these parameters (p < 0.01). In PA animals, a large number of apoptotic cells was found in the GCL. This number was significantly reduced by treatment with the small molecule (p < 0.0001). In a similar way, the thickness of the inner retina and the intensity of GFAP immunoreactivity (gliosis) increased in PA retinas (p < 0.0001). These parameters were corrected by the administration of zr17-2 (p < 0.0001). Furthermore, injection of the small molecule in the absence of PA did not modify the ERG nor the morphological parameters studied, suggesting a lack of toxicity. Discussion: In conclusion, our results indicate that a single s.c. injection of zr17-2 in asphyctic neonates may provide a novel and efficacious method to prevent the visual sequelae of PA.

A hypothermia mimetic molecule (zr17-2) reduces ganglion cell death and electroretinogram distortion in a rat model of intraorbital optic nerve crush (IONC).

Introduction: Ocular and periocular traumatisms may result in loss of vision. Our previous work showed that therapeutic hypothermia prevents retinal damage caused by traumatic neuropathy. We also generated and characterized small molecules that elicit the beneficial effects of hypothermia at normal body temperature. Here we investigate whether one of these mimetic molecules, zr17-2, is able to preserve the function of eyes exposed to trauma. Methods: Intraorbital optic nerve crush (IONC) or sham manipulation was applied to Sprague-Dawley rats. One hour after surgery, 5.0 µl of 330 nmol/L zr17-2 or PBS, as vehicle, were injected in the vitreum of treated animals. Electroretinograms were performed 21 days after surgery and a- and b-wave amplitude, as well as oscillatory potentials (OP), were calculated. Some animals were sacrificed 6 days after surgery for TUNEL analysis. All animal experiments were approved by the local ethics board. Results: Our previous studies showed that zr17-2 does not cross the blood-ocular barrier, thus preventing systemic treatment. Here we show that intravitreal injection of zr17-2 results in a very significant prevention of retinal damage, providing preclinical support for its pharmacological use in ocular conditions. As previously reported, IONC resulted in a drastic reduction in the amplitude of the b-wave (p < 0.0001) and OPs (p < 0.05), a large decrease in the number of RGCs (p < 0.0001), and a large increase in the number of apoptotic cells in the GCL and the INL (p < 0.0001). Interestingly, injection of zr17-2 largely prevented all these parameters, in a very similar pattern to that elicited by therapeutic hypothermia. The small molecule was also able to reduce oxidative stress-induced retinal cell death in vitro. Discussion: In summary, we have shown that intravitreal injection of the hypothermia mimetic, zr17-2, significantly reduces the morphological and electrophysiological consequences of ocular traumatism and may represent a new treatment option for this cause of visual loss.

Adenosine A2A Receptor: A New Neuroprotective Target in Light-Induced Retinal Degeneration.

Continuous illumination induces the degeneration of photoreceptors. This animal model of light-induced retinal degeneration resembles many characteristics of human degenerative diseases of the outer retina, such as age-related macular degeneration. This work aimed to evaluate the potential neuroprotective effect of the modulation of adenosine A2A receptor in the model of light-induced retinal degeneration. Sprague-Dawley rats were intravitreally injected in the right eye with either CGS 21680, an adenosine A2A receptor agonist, or SCH 58261, an adenosine A2A receptor antagonist. Contralateral eyes were injected with respective vehicles as control. Then, rats were subjected to continuous illumination (12,000 lux) for 24 h. Retinas were processed by glial fibrillary acidic protein (GFAP) immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique, Western blotting (WB), and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Another group of rats was subjected to functional studies by electroretinography. Animals treated with CGS21680 showed a significant increase of apoptotic nuclei in the outer nuclear layer and a significant increase of GFAP immunoreactive area of the retinas but did not alter WB nor electroretinography results. qRT-PCR showed that CGS 21680 significantly increased the expression of interleukin-1ß. On the opposite, SCH 58261 significantly decreased apoptotic nuclei in the outer nuclear layer and GFAP immunoreactive area of the retinas. It also significantly decreased GFAP and activated caspase-3 levels as measured by WB and preserved retinal function, as treated eyes showed significantly greater amplitudes of a- and b-waves and oscillatory potentials. qRT-PCR revealed that SCH 58261 significantly decreased the expression of tumor necrosis factor-α. These results show that the blockade of the A2A receptor before the start of the pathogenic process is neuroprotective, as it prevents light-induced retinal damage. The use of A2A receptor antagonists deserves to be evaluated in retinal degenerative diseases.

CB1 Cannabinoid Receptor is a Target for Neuroprotection in Light Induced Retinal Degeneration.

In the last few years, an increasing interest in the neuroprotective effect of cannabinoids has taken place. The aim of the present work was to study the effects of modulating cannabinoid receptor 1 (CB1) in the context of light induced retinal degeneration (LIRD), using an animal model that resembles many characteristics of human age-related macular degeneration (AMD) and other degenerative diseases of the outer retina. Sprague Dawley rats (n = 28) were intravitreally injected in the right eye with either a CB1 agonist (ACEA), or an antagonist (AM251). Contralateral eyes were injected with respective vehicles as controls. Then, rats were subjected to continuous illumination (12,000 lux) for 24 h. Retinas from 28 animals were processed by GFAP-immunohistochemistry (IHC), TUNEL technique, Western blotting (WB), or qRT-PCR. ACEA-treated retinas showed a significantly lower number of apoptotic nuclei in the outer nuclear layer (ONL), lower levels of activated Caspase-3 by WB, and lower levels of glial reactivity by both GFAP-IHC and WB. qRT-PCR revealed that ACEA significantly decreased the expression of Bcl-2 and CYP1A1. Conversely, AM251-treated retinas showed a higher number of apoptotic nuclei in the ONL, higher levels of activated Caspase-3 by WB, and higher levels of glial reactivity as determined by GFAP-IHC and WB. AM251 increased the expression of Bcl-2, Bad, Bax, Aryl hydrocarbon Receptor (AhR), GFAP, and TNFα. In summary, the stimulation of the CB1 receptor, previous to the start of the pathogenic process, improved the survival of photoreceptors exposed to LIRD. The modulation of CB1 activity may be used as a neuroprotective strategy in retinal degeneration and deserves further studies.

Expresión de proteínas inducibles por frío en la médula espinal de rata sometida a hipotermia sistémica / Expression of cold-inducible proteins in rat spinal cord subjected to systemic hypothermia

Introducción: La lesión traumática de la médula espinal es la principal causa de discapacidad motora en el mundo, y representa una prioridad para la Organización Mundial de la Salud. Se estudió, a nivel estructural y bioquímico, el efecto de la hipotermia sobre la expresión de la CIRBP (proteína activada por frío) en el asta anterior de la médula de ratas Sprague-Dawley albinas macho de 60 días, planteándola como terapéutica posible. Materiales y Métodos:Se dividió a 24 ratas en dos grupos: normotermia a 24 °C (n = 6) e hipotermia a 8 °C (n = 18), durante 180 min, sacrificadas a las 12, 24 y 48 h después del tratamiento. Se utilizó Western blot e inmunohistoquímica para la CIRBP. Resultados:Se observó un aumento progresivo de la expresión de la CIRBP de 12 a 48 h en las motoneuronas del asta anterior. Los valores fueron estadísticamente significativos entre los grupos de 24 h y 48 h comparados con los de los controles. Conclusiones: Este modelo experimental resultó eficaz, accesible y económico para generar hipotermia sistémica y abre un abanico de estrategias terapéuticas. El aumento en la expresión de las proteínas inducibles por frío en la médula espinal de ratas permite, por primera vez, estudiar el beneficio que aporta la hipotermia a nivel molecular, lo que resulta de suma importancia para estudios de terapéuticas en las lesiones medulares. Nivel de Evidencia: I

Introduction: Traumatic spinal cord injury is the main cause of motor disability in developed and underdeveloped countries, being a priority interest to the WHO. The effect of hypothermia on the expression of CIRBP (cold-activated protein) in the anterior grey column of 60-day-old male albino Sprague-Dawley rats was studied at the structural and biochemical levels and proposed as a possible therapeutic approach. Materials and Methods: 24 rats were randomly divided into two groups; normothermia (n = 6), at 24° C, and hypothermia, (n = 18) at 8° C for 180 minutes and euthanized at 12, 24, and 48 h post-treatment. Western blot and immunohistochemistry for CIRBP were used. Results: A progressive increase in the expression of CIRBP was observed from 12 to 48 hours, with statistically significant values after 24 and 48 hours compared to controls. Conclusion: This experimental model demonstrated efficacy, accessibility, and economy to generate systemic hypothermia, which provides a novel range of therapeutic strategies. The increase in the expression of cold-inducible proteins in the rats' spinal cords allows us to study the benefit of hypothermia at the molecular level for the first time, being of utmost importance for therapeutic studies in spinal cord injuries. Level of Evidence: I

Modelo de hipotermia experimental en murinos para estudios de lesión medular / Experimental hypothermia murine model for spinal cord injury studies

Introducción: Los ensayos de hipotermia sistémica en murinos son costosos, debido a la complejidad de los sistemas. El objetivo de este estudio fue evaluar si el modelo de hipotermia sistémica exógena utilizado en nuestro laboratorio para la hipotermia ocular es útil para reducir significativamente la temperatura de la médula espinal en ratas adultas. Materiales y métodos: Se utilizaron 36 ratas Sprague-Dawley albinas macho de 60 días, distribuidas en dos grupos: grupo normotermia a 24 °C (n = 18) y grupo hipotermia (n = 18) en cámara fría a 8 °C durante 180 minutos. Resultados: La temperatura rectal promedio fue de 37,71 ± 0,572 °C en el grupo normotermia y 34,03 ± 0,250 °C en el grupo hipotermia (p <0,0001). La temperatura medular promedio fue de 38,8 ± 0,468 °C en el grupo normotermia y de 36,4 ± 0,290 °C en el grupo hipotermia (p <0,0001). Conclusiones: El uso de hipotermia sistémica en ratas de laboratorio parece ser un método prometedor para evaluar los mecanismos fisiológicos y patológicos que se desencadenan en la médula espinal. La exposición al frío en cámara genera hipotermia medular significativa en ratas adultas. Los resultados sugieren que podría ser un modelo adecuado de hipotermia medular de bajo costo. Nivel de Evidencia: III

Given the complexity of hypothermal trial systems in murines, they are expensive. Our objective was to evaluate if the exogenous hypothermal model used in our laboratory for ocular hypothermia was useful for a significant reduction in medullar spine temperature in adult murines. Materials and methods: 36 60-day-old adult male Sprague-Dawley rats were used. They were separated into two groups: a normal temperature group at 24 °C (n=18) and a hypothermia group in a cold chamber at 8 °C for 180 minutes (n=18). Results: The mean rectal temperature was 37.71 °C ± 0.572 in the normothermia group and 34.03°C ± 0.250 in the hypothermia group (p <0.0001). The mean medullar temperature was 38.8 ± 0.468 °C in the normothermia group and 36.4 ± 0.290 °C in the hypothermia group (p <0.0001). Conclusion: Using systematic hypothermia in lab rats seems to be promising to evaluate physiologic and pathological mechanisms triggered in the medullar spine. Exposure to cold in the external chamber produces significant medullar hypothermia in adult rats. Results suggest this might be an adequate and inexpensive medullar hypothermal model. Level of Evidence: III

Hypothermic Shock Applied After Perinatal Asphyxia Prevents Retinal Damage in Rats.

Perinatal asphyxia (PA) can cause retinopathy and different degrees of visual loss, including total blindness. In a rat model of PA, we have previously shown a protective effect of hypothermia on the retina when applied simultaneously with the hypoxic insult. In the present work, we evaluated the possible protective effect of hypothermia on the retina of PA rats when applied immediately after delivery. Four experimental groups were studied: Rats born naturally as controls (CTL), animals that were exposed to PA for 20 min at 37°C (PA), animals exposed to PA for 20 min at 15°C (HYP), and animals that were exposed to PA for 20 min at 37°C and, immediately after birth, kept for 15 min at 8°C (HYP-PA). To evaluate the integrity of the visual pathway, animals were subjected to electroretinography at 45 days of age. Molecular (real time PCR) and histological (immunohistochemistry, immunofluorescence, TUNEL assay) techniques were applied to the eyes of all experimental groups collected at 6, 12, 24, and 48 h, and 6 days after birth. PA resulted in a significant reduction in the amplitude of the a- and b-wave and oscillatory potentials (OP) of the electroretinogram. All animals treated with hypothermia had a significant correction of the a-wave and OP, but the b-wave was fully corrected in the HYP group but only partially in the HYP-PA group. The number of TUNEL-positive cells increased sharply in the ganglion cell layer of the PA animals and this increase was significantly prevented by both hypothermia treatments. Expression of the cold-shock proteins, cold-inducible RNA binding protein (CIRP) and RNA binding motif protein 3 (RBM3), was undetectable in retinas of the CTL and PA groups, but they were highly expressed in ganglion neurons and cells of the inner nuclear layer of the HYP and HYP-PA groups. In conclusion, our results suggest that a post-partum hypothermic shock could represent a useful and affordable method to prevent asphyxia-related vision disabling sequelae.

Methylene Blue Prevents Retinal Damage Caused by Perinatal Asphyxia in the Rat.

Perinatal asphyxia (PA) is responsible for a large proportion of neonatal deaths and numerous neurological sequelae, including visual dysfunction and blindness. In PA, the retina is exposed to ischemia/reoxygenation, which results in nitric oxide (NO) overproduction and neurotoxicity. We hypothesized that methylene blue (MB), a guanylyl cyclase inhibitor, and free-radical scavenger currently used in the clinic, may block this pathway and prevent PA-induced retinal degeneration. Male rat pups were subjected to an experimental model of PA. Four groups were studied: normally delivered (CTL), normally delivered treated with 2 mg Kg-1 MB (MB), exposed to PA for 20 min at 37°C (PA), and exposed to PA and, then, treated with MB (PA-MB). Scotopic electroretinography performed 45 days after birth showed that PA animals had significant defects in the a- and b-waves and oscillatory potentials (OP). The same animals presented a significant increase in the thickness of the inner retina and a large number of TUNEL-positive cells. All these physiological and morphological parameters were significantly prevented by the treatment with MB. Gene expression analysis demonstrated significant increases in iNOS, MMP9, and VEGF in the eyes of PA animals, which were prevented by MB treatment. In conclusion, MB regulates key players of inflammation, matrix remodeling, gliosis, and angiogenesis in the eye and could be used as a treatment to prevent the deleterious visual consequences of PA. Given its safety profile and low cost, MB may be used clinically in places where alternative treatments may be unavailable.

Statistical differences resulting from selection of stable reference genes after hypoxia and hypothermia in the neonatal rat brain.

Real-time reverse transcription PCR (qPCR) normalized to an internal reference gene (RG), is a frequently used method for quantifying gene expression changes in neuroscience. Although RG expression is assumed to be constant independent of physiological or experimental conditions, several studies have shown that commonly used RGs are not expressed stably. The use of unstable RGs has a profound effect on the conclusions drawn from studies on gene expression, and almost universally results in spurious estimation of target gene expression. Approaches aimed at selecting and validating RGs often make use of different statistical methods, which may lead to conflicting results. Based on published RG validation studies involving hypoxia the present study evaluates the expression of 5 candidate RGs (Actb, Pgk1, Sdha, Gapdh, Rnu6b) as a function of hypoxia exposure and hypothermic treatment in the neonatal rat cerebral cortex-in order to identify RGs that are stably expressed under these experimental conditions-using several statistical approaches that have been proposed to validate RGs. In doing so, we first analyzed RG ranking stability proposed by several widely used statistical methods and related tools, i.e. the Coefficient of Variation (CV) analysis, GeNorm, NormFinder, BestKeeper, and the ΔCt method. Using the Geometric mean rank, Pgk1 was identified as the most stable gene. Subsequently, we compared RG expression patterns between the various experimental groups. We found that these statistical methods, next to producing different rankings per se, all ranked RGs displaying significant differences in expression levels between groups as the most stable RG. As a consequence, when assessing the impact of RG selection on target gene expression quantification, substantial differences in target gene expression profiles were observed. Altogether, by assessing mRNA expression profiles within the neonatal rat brain cortex in hypoxia and hypothermia as a showcase, this study underlines the importance of further validating RGs for each individual experimental paradigm, considering the limitations of the statistical methods used for this aim.

The expression of adenosine receptors changes throughout light induced retinal degeneration in the rat.

The modulation of adenosine receptors, A1 (A1R) and A2A (A2AR), is neuroprotective in different models of retinal injury. In order to understand the processes underlying retinal degeneration, we studied the expression of adenosine receptors in the retinas of control and continuously illuminated (CI) rats by qRT-PCR, Western blot (WB) and immunohistochemistry (IHC). Significant increases of A1R, A2AR, and A2BR mRNAs at 1, 5, and 7 days of CI (P < 0.0001) were observed by qRT-PCR. Also, a significant increase of A3R mRNA was detected after 5 and 7 days of CI. WB studies showed a significant rise of A1R on day 1 of CI and on days 5 and 7 (P < 0.0001), while A2AR increase was seen from 2 days of CI on (P < 0.001). After 1 day of CI, A1R immunoreactivity (A1R-IR) increased in ganglion cell layer, inner nuclear layer, and in both the outer and inner plexiform layers. After 2 days of CI, the A1R-IR went back to control levels. After 5 days of CI, a second rise in A1R, which persisted until 7 days of CI, was measured (P < 0.0001). A significant rise of A2aR immunoreactivity was also observed at day 2 of CI at GCL and INL and subsided at days 5 and 7 (P < 0.0001). The observed up-regulation of A1R after 1 day of CI, corresponds with the peak of oxidative stress; while the rise of A2aR at day 2 of CI, coincides with the massive apoptosis of photoreceptors. We postulate that an early modulation of adenosine receptors could delay or prevent the degeneration of photoreceptors.

Adenosine A1 receptor: A neuroprotective target in light induced retinal degeneration.

Light induced retinal degeneration (LIRD) is a useful model that resembles human retinal degenerative diseases. The modulation of adenosine A1 receptor is neuroprotective in different models of retinal injury. The aim of this work was to evaluate the potential neuroprotective effect of the modulation of A1 receptor in LIRD. The eyes of rats intravitreally injected with N6-cyclopentyladenosine (CPA), an A1 agonist, which were later subjected to continuous illumination (CI) for 24 h, showed retinas with a lower number of apoptotic nuclei and a decrease of Glial Fibrillary Acidic Protein (GFAP) immunoreactive area than controls. Lower levels of activated Caspase 3 and GFAP were demonstrated by Western Blot (WB) in treated animals. Also a decrease of iNOS, TNFα and GFAP mRNA was demonstrated by RT-PCR. A decrease of Iba 1+/MHC-II+ reactive microglial cells was shown by immunohistochemistry. Electroretinograms (ERG) showed higher amplitudes of a-wave, b-wave and oscillatory potentials after CI compared to controls. Conversely, the eyes of rats intravitreally injected with dipropylcyclopentylxanthine (DPCPX), an A1 antagonist, and subjected to CI for 24 h, showed retinas with a higher number of apoptotic nuclei and an increase of GFAP immunoreactive area compared to controls. Also, higher levels of activated Caspase 3 and GFAP were demonstrated by Western Blot. The mRNA levels of iNOS, nNOS and inflammatory cytokines (IL-1ß and TNFα) were not modified by DPCPX treatment. An increase of Iba 1+/MHC-II+ reactive microglial cells was shown by immunohistochemistry. ERG showed that the amplitudes of a-wave, b-wave, and oscillatory potentials after CI were similar to control values. A single pharmacological intervention prior illumination stress was able to swing retinal fate in opposite directions: CPA was neuroprotective, while DPCPX worsened retinal damage. In summary, A1 receptor agonism is a plausible neuroprotective strategy in LIRD.

Hypothermia Prevents Retinal Damage Generated by Optic Nerve Trauma in the Rat.

Ocular and periocular traumatisms may result in loss of vision. Hypothermia provides a beneficial intervention for brain and heart conditions and, here, we study whether hypothermia can prevent retinal damage caused by traumatic neuropathy. Intraorbital optic nerve crush (IONC) or sham manipulation was applied to male rats. Some animals were subjected to hypothermia (8 °C) for 3 h following surgery. Thirty days later, animals were subjected to electroretinography and behavioral tests. IONC treatment resulted in amplitude reduction of the b-wave and oscillatory potentials of the electroretinogram, whereas the hypothermic treatment significantly (p < 0.05) reversed this process. Using a descending method of limits in a two-choice visual task apparatus, we demonstrated that hypothermia significantly (p < 0.001) preserved visual acuity. Furthermore, IONC-treated rats had a lower (p < 0.0001) number of retinal ganglion cells and a higher (p < 0.0001) number of TUNEL-positive cells than sham-operated controls. These numbers were significantly (p < 0.0001) corrected by hypothermic treatment. There was a significant (p < 0.001) increase of RNA-binding motif protein 3 (RBM3) and of BCL2 (p < 0.01) mRNA expression in the eyes exposed to hypothermia. In conclusion, hypothermia constitutes an efficacious treatment for traumatic vision-impairing conditions, and the cold-shock protein pathway may be involved in mediating the beneficial effects shown in the retina.

Concerted regulation of renal plasma flow and glomerular filtration rate by renal dopamine and NOS I in rats on high salt intake.

Under high sodium intake renal dopamine (DA) increases while NOS I expression in macula densa cells (MD) decreases. To explore whether renal DA and NOS I, linked to natriuresis and to the stability of the tubuloglomerular feedback, respectively, act in concert to regulate renal plasma flow (RPF) and glomerular filtration rate (GFR). Male Wistar rats were studied under a normal sodium intake (NS, NaCl 0.24%) or a high sodium intake (HS, NaCl 1% in drinking water) during the 5 days of the study. For the last two days, the specific D1-like receptor antagonist SCH 23390 (1 mg kg bwt-1 day-1, sc) or a vehicle was administered. HS intake increased natriuresis, diuresis, and urinary DA while it decreased cortical NOS I expression (P < 0.05 vs. NS), Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in MD (P < 0.001 vs. NS) and cortical nitrates+nitrites (NOx) production (NS 2.04 ± 0.22 vs. HS 1.28 ± 0.10 nmol mg protein-1, P < 0.01). Treatment with SCH 23390 to rats on HS sharply decreased hydroelectrolyte excretion (P < 0.001 vs. HS) while NOS I expression, NADPH-d activity and NOx production increased (P < 0.05 vs. HS for NOS I and P < 0.001 vs. HS for NADPH-d and NOx). SCH 23390 increased RPF and GFR in HS rats (P < 0.01 HS+SCH vs. HS). It did not cause variations in NS rats. Results indicate that when NS intake is shifted to a prolonged high sodium intake, renal DA through the D1R, and NOS I in MD cells act in concert to regulate RPF and GFR to stabilize the delivery of NaCl to the distal nephron.

Cold Shock Proteins Are Expressed in the Retina Following Exposure to Low Temperatures.

Hypothermia has been proposed as a therapeutic intervention for some retinal conditions, including ischemic insults. Cold exposure elevates expression of cold-shock proteins (CSP), including RNA-binding motif protein 3 (RBM3) and cold inducible RNA-binding protein (CIRP), but their presence in mammalian retina is so far unknown. Here we show the effects of hypothermia on the expression of these CSPs in retina-derived cell lines and in the retina of newborn and adult rats. Two cell lines of retinal origin, R28 and mRPE, were exposed to 32°C for different time periods and CSP expression was measured by qRT-PCR and Western blotting. Neonatal and adult Sprague-Dawley rats were exposed to a cold environment (8°C) and expression of CSPs in their retinas was studied by Western blotting, multiple inmunofluorescence, and confocal microscopy. RBM3 expression was upregulated by cold in both R28 and mRPE cells in a time-dependent fashion. On the other hand, CIRP was upregulated in R28 cells but not in mRPE. In vivo, expression of CSPs was negligible in the retina of newborn and adult rats kept at room temperature (24°C). Exposure to a cold environment elicited a strong expression of both proteins, especially in retinal pigment epithelium cells, photoreceptors, bipolar, amacrine and horizontal cells, Müller cells, and ganglion cells. In conclusion, CSP expression rapidly rises in the mammalian retina following exposure to hypothermia in a cell type-specific pattern. This observation may be at the basis of the molecular mechanism by which hypothermia exerts its therapeutic effects in the retina.

Methylene blue prevents retinal damage in an experimental model of ischemic proliferative retinopathy.

Perinatal asphyxia induces retinal lesions, generating ischemic proliferative retinopathy, which may result in blindness. Previously, we showed that the nitrergic system was involved in the physiopathology of perinatal asphyxia. Here we analyze the application of methylene blue, a well-known soluble guanylate cyclase inhibitor, as a therapeutic strategy to prevent retinopathy. Male rats (n = 28 per group) were treated in different ways: 1) control group comprised born-to-term animals; 2) methylene blue group comprised animals born from pregnant rats treated with methylene blue (2 mg/kg) 30 and 5 min before delivery; 3) perinatal asphyxia (PA) group comprised rats exposed to perinatal asphyxia (20 min at 37°C); and 4) methylene blue-PA group comprised animals born from pregnant rats treated with methylene blue (2 mg/kg) 30 and 5 min before delivery, and then the pups were subjected to PA as above. For molecular studies, mRNA was obtained at different times after asphyxia, and tissue was collected at 30 days for morphological and biochemical analysis. Perinatal asphyxia produced significant gliosis, angiogenesis, and thickening of the inner retina. Methylene blue treatment reduced these parameters. Perinatal asphyxia resulted in a significant elevation of the nitrergic system as shown by NO synthase (NOS) activity assays, Western blotting, and (immuno)histochemistry for the neuronal isoform of NOS and NADPH-diaphorase activity. All these parameters were also normalized by the treatment. In addition, methylene blue induced the upregulation of the anti-angiogenic peptide, pigment epithelium-derived factor. Application of methylene blue reduced morphological and biochemical parameters of retinopathy. This finding suggests the use of methylene blue as a new treatment to prevent or decrease retinal damage in the context of ischemic proliferative retinopathy.

Hypothermia prevents gliosis and angiogenesis development in an experimental model of ischemic proliferative retinopathy.

PURPOSE: To develop a time course study of vascularization and glial response to perinatal asphyxia in hypoxic-ischemic animals, and to evaluate hypothermia as possible protective treatment. METHODS: We used retinas of 7-, 15-, 21-, and 30-day-old male Sprague-Dawley rats that were exposed to perinatal asphyxia at either 37°C (PA) or 15°C (HYP). Born to term animals were used as controls (CTL). We evaluated the thickness of the most inner layers of the retina (IR), including internal limiting membrane, the retinal nerve fiber layer, and the ganglion cell layer; and studied glial development, neovascularization, adrenomedullin (AM), and VEGF by immunohistochemistry, immunofluorescence, and Western blot. RESULTS: A significant increment in IR thickness was observed in the PA group from postnatal day (PND) 15 on. This alteration was concordant with an increased number of new vessels and increased GFAP expression. The immunolocalization of GFAP in the internal limiting membrane and perivascular glia of the IR and in the inner processes of Müller cells was coexpressed with AM, which was also significantly increased from PND7 in PA animals. In addition, VEGF expression was immunolocalized in cells of the ganglion cell layer of the IR and this expression significantly increased in the PA group from PND15 on. The retinas of the HYP group did not show differences when compared with CTL at any age. CONCLUSIONS: This work demonstrates that aberrant angiogenesis and exacerbated gliosis seem to be responsible for the increased thickness of the inner retina as a consequence of perinatal asphyxia, and that hypothermia is able to prevent these alterations.

Epigenetic modification of liver mitochondrial DNA is associated with histological severity of nonalcoholic fatty liver disease.

OBJECTIVE & DESIGN: Nonalcoholic fatty liver disease (NAFLD) is a clinical condition that refers to progressive histological changes ranging from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH). We evaluated the status of cytosine methylation (5mC) of liver mitochondrial DNA (mtDNA) in selected regions of the mtDNA genome, such as D-loop control region, and mitochondrially encoded NADH dehydrogenase 6 (MT-ND6) and cytochrome C oxidase I (MT-CO1), to contrast the hypothesis that epigenetic modifications play a role in the phenotypic switching from SS to NASH. METHODS: We studied liver biopsies obtained from patients with NAFLD in a case-control design; 45 patients and 18 near-normal liver-histology subjects. RESULTS: MT-ND6 methylation was higher in the liver of NASH than SS patients (p < 0.04) and MT-ND6 methylated DNA/unmethylated DNA ratio was significantly associated with NAFLD activity score (p < 0.02). Liver MT-ND6 mRNA expression was significantly decreased in NASH patients (0.26 ± 0.30) versus SS (0.74 ± 0.48), p < 0.003, and the protein level was also diminished. The status of liver MT-ND6 methylation in NASH group was inversely correlated with the level of regular physical activity (R = -0.54, p < 0.02). Hepatic methylation levels of D-Loop and MT-CO1 were not associated with the disease severity. DNA (cytosine-5) methyltransferase 1 was significantly upregulated in NASH patients (p < 0.002). Ultrastructural evaluation showed that NASH is associated with mitochondrial defects and peroxisome proliferation. CONCLUSION: Hepatic methylation and transcriptional activity of the MT-ND6 are associated with the histological severity of NAFLD. Epigenetic changes of mtDNA are potentially reversible by interventional programs, as physical activity could modulate the methylation status of MT-ND6.

Hypothermia prevents nitric oxide system changes in retina induced by severe perinatal asphyxia.

One-third of asphyctic neonates develop long-term neurological injuries, including several degrees of ischemic proliferative retinopathy (IPR) such as retinopathy of prematurity (ROP). Given that the retina is altered by perinatal asphyxia, our aim was to study the effects of nitric oxide (NO) in the retina in order to analyze its impact on the retinal injury. Application of hypothermia was evaluated as preventive treatment. Sprague-Dawley rats were subjected to perinatal asphyxia [either at 37°C (PA group) or at 15°C (HYP group)]. Full-term rats were used as controls (CTL). A significantly increased activity of both constitutive NO synthase (nNOS, Ca(2+)-dependent) and inducible NO synthase (iNOS, Ca(2+)-independent) was observed in PA retinas from 21 days old up to 60 days old with respect to age-matched CTL, with a significant increase along the time course in the PA. nNOS was immunolocalized at amacrine, horizontal, and ganglion cells of the PA group, with a significant increase in relative optical density (R.O.D.), cellular area, and number of cells. iNOS immunoreactivity was observed in the inner nuclear layer and in the internal Müller cell processes of PA, with a significant increase in R.O.D. and colocalizing with GFAP in the 60-day-old PA group. Six nitrated protein species were increased in retinas from PA rats. Nitrotyrosine immunoreactivity showed a localization similar to that of iNOS, with increased R.O.D. in the PA group and colocalization with GFAP in 60-day-old animals. HYP prevented all the changes observed in PA rats. Although the NO system displays changes induced by hypoxia-ischemia, hypothermia application shows a strong protective effect.

Hypothermia prevents the development of ischemic proliferative retinopathy induced by severe perinatal asphyxia.

Obstetric complications, such as perinatal asphyxia, may cause retinal injuries as retinopathy of prematurity (ROP), a type of ischemic proliferative retinopathy. Up to date there are no appropriate experimental models for studying the long-term sequels of this disease. In the present work, we present an experimental model of perinatal asphyxia which shows structural and ultrastructural retinal alterations at the most inner layers of the retina, such as neurodegeneration, development of neoformed vessels and glial reaction, which are compatible with the histopathological description of ROP. Besides, the application of hypothermia during perinatal asphyxia showed effective results preventing cellular and morphological alterations. This study may contribute to the development of therapies in order to either ameliorate or prevent retinal damage. In this manner, hypothermia may improve life quality and decrease medical, family and social costs of these avoidable causes of blindness.

Nitric oxide system alteration at spinal cord as a result of perinatal asphyxia is involved in behavioral disabilities: hypothermia as preventive treatment.

Perinatal asphyxia (PA) is able to induce sequelae such as spinal spasticity. Previously, we demonstrated hypothermia as a neuroprotective treatment against cell degeneration triggered by increased nitric oxide (NO) release. Because spinal motoneurons are implicated in spasticity, our aim was to analyze the involvement of NO system at cervical and lumbar motoneurons after PA as well as the application of hypothermia as treatment. PA was performed by immersion of both uterine horns containing full-term fetuses in a water bath at 37 degrees C for 19 or 20 min (PA19 or PA20) or at 15 degrees C for 20 min (hypothermia during PA-HYP). Some randomly chosen PA20 rats were immediately exposed for 5 min over grain ice (hypothermia after PA-HPA). Full-term vaginally delivered rats were used as control (CTL). We analyzed NO synthase (NOS) activity, expression and localization by nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) reactivity, inducible and neuronal NOS (iNOS and nNOS) by immunohistochemistry, and protein nitrotyrosilation state. We observed an increased NOS activity at cervical spinal cord of 60-day-old PA20 rats, with increased NADPH-d, iNOS, and nitrotyrosine expression in cervical motoneurons and increased NADPH-d in neurons of layer X. Lumbar neurons were not altered. Hypothermia was able to maintain CTL values. Also, we observed decreased forelimb motor potency in the PA20 group, which could be attributed to changes at cervical motoneurons. This study shows that PA can induce spasticity produced by alterations in the NO system of the cervical spinal cord. Moreover, this situation can be prevented by perinatal hypothermia.