Streptozotocin-Induced Diabetic Rats Showed a Differential Glycine Receptor Expression in the Spinal Cord: A GlyR Role in Diabetic Neuropathy.

In the spinal cord, attenuation of the inhibitory action of glycine is related to an increase in both inflammatory and diabetic neuropathic pain; however, the glycine receptor involvement in diabetic neuropathy has not been reported. We determined the expression of the glycine receptor subunits (α1-α3 and ß) in streptozotocin-induced diabetic Long-Evans rats by qPCR and Western blot. The total mRNA and protein expression (whole spinal cord homogenate) of the α1, α3, and ß subunits did not change during diabetes; however, the α2 subunit mRNA, but not the protein, was overexpressed 45 days after diabetes induction. By contrast, the synaptic expression of the α1 and α2 subunits decreased in all the studied stages of diabetes, but that of the α3 subunit increased on day 45 after diabetes induction. Intradermal capsaicin produced higher paw-licking behavior in the streptozotocin-induced diabetic rats than in the control animals. In addition, the nocifensive response was higher at 45 days than at 20 days. During diabetes, the expression of the glycine receptor was altered in the spinal cord, which strongly suggests its involvement in diabetic neuropathy.

Retinal Nrf2 expression in normal and early streptozotocin-diabetic rats.

Diabetic retinopathy is the most common cause of vision loss among diabetic patients. Although hyperglycemia produces retinal oxidative stress in long-standing diabetes, the pathogenesis mechanism is unknown. The Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a central role in cell responses against oxidative damage. We used adult Long Evans rats where diabetes was induced by streptozotocin. Normal and treated rats were sacrificed at 7, 20, and 45 days after streptozotocin injection. We analyzed Nrf2 and Keap1 expression in retinal homogenates, cytoplasmic, and nuclear retinal fractions. Normal retina showed Nrf2 expression in all retina nuclear layers. We found a transitory decrease of Nrf2 mRNA and protein expression at 7 and 20 days after the streptozotocin injection that recovered later on: moreover, the protein level increased after 45 days. Keap1 immunoprecipitation revealed similar levels as Nrf2 in normal and diabetic rat retinas, indicating that the diabetic condition did not lead to dissociation of the Keap1-Nrf2 complex. Indeed, glutathione levels and superoxide dismutase activity were not altered in the treated rat retinas. These results do not support oxidative stress in the retina shortly after diabetes induction.

Artificial Neural Networks Predict 30-Day Mortality After Hip Fracture: Insights From Machine Learning.

OBJECTIVES: Accurately stratifying patients in the preoperative period according to mortality risk informs treatment considerations and guides adjustments to bundled reimbursements. We developed and compared three machine learning models to determine which best predicts 30-day mortality after hip fracture. METHODS: The 2016 to 2017 National Surgical Quality Improvement Program for hip fracture (AO/OTA 31-A-B-C) procedure-targeted data were analyzed. Three models-artificial neural network, naive Bayes, and logistic regression-were trained and tested using independent variables selected via backward variable selection. The data were split into 80% training and 20% test sets. Predictive accuracy between models was evaluated using area under the curve receiver operating characteristics. Odds ratios were determined using multivariate logistic regression with P < 0.05 for significance. RESULTS: The study cohort included 19,835 patients (69.3% women). The 30-day mortality rate was 5.3%. In total, 47 independent patient variables were identified to train the testing models. Area under the curve receiver operating characteristics for 30-day mortality was highest for artificial neural network (0.92), followed by the logistic regression (0.87) and naive Bayes models (0.83). DISCUSSION: Machine learning is an emerging approach to develop accurate risk calculators that account for the weighted interactions between variables. In this study, we developed and tested a neural network model that was highly accurate for predicting 30-day mortality after hip fracture. This was superior to the naive Bayes and logistic regression models. The role of machine learning models to predict orthopaedic outcomes merits further development and prospective validation but shows strong promise for positively impacting patient care.

Distal Femur Replacement Versus Surgical Fixation for the Treatment of Geriatric Distal Femur Fractures: A Systematic Review.

OBJECTIVES: The management of geriatric distal femur fractures is controversial, and both primary distal femur replacement (DFR) and surgical fixation (SF) are viable treatment options. The purpose of this study was to compare patient outcomes after these treatment strategies. DATA SOURCES: PubMed, Embase, and Cochrane databases were searched for English language articles up to April 24, 2020, identifying 2129 papers. STUDY SELECTION: Studies evaluating complications in elderly patients treated for distal femur fractures with either immediate DFR or SF were included. Studies with mean patient age <55 years, nontraumatic indications for DFR, or SF with nonlocking plates were excluded. DATA EXTRACTION: Two studies provided Level II or III evidence, whereas the remaining 28 studies provided Level IV evidence. Studies were formally evaluated for methodological quality using established criteria. Treatment failure between groups was compared using an incidence rate ratio. DATA SYNTHESIS: Treatment failure was defined for both SF and arthroplasty as complications requiring a major reoperation for reasons such as mechanical failure, nonunion, deep infection, aseptic loosening, or extensor mechanism disruption. There were no significant differences in complication rates or knee range of motion between SF and DFR. CONCLUSIONS: SF and DFR for the treatment of geriatric distal femur fractures demonstrate similar overall complication rates. Given the available evidence, no strong conclusions on the comparative effectiveness between the 2 treatments can be definitively made. More rigorous prospective research comparing SF vs. DFR to treat acute geriatric distal femur fractures is warranted. LEVEL OF EVIDENCE: Therapeutic Level IV. See instructions for authors for a complete description of levels of evidence.

Low-grade chronic inflammation is attenuated by exercise training in obese adults through down-regulation of ASC gene in peripheral blood: a pilot study.

BACKGROUND: Obesity is characterized by low-grade chronic inflammation and an excess of adipose tissue. The ASC gene encodes a protein that is part of the NLRP3 inflammasome, a cytosolic multiprotein complex that is associated with inflammation and metabolic alterations. To our knowledge, there is no evidence regarding ASC gene activity in obese adults in response to lifestyle modifications. PURPOSE: To evaluate the effect of hypocaloric diet and moderate-intensity structured exercise intervention on ASC gene expression and inflammatory markers in obese adults. METHODS: Thirty-seven obese individuals aged 25 to 50 years were randomized to the hypocaloric diet exercise group or hypocaloric diet group. The participants underwent a 4-month follow-up. Electrical bioimpedance was used for body composition analysis. Biochemical data were analyzed by dry chemistry and insulin levels by ELISA. ASC gene expression from peripheral blood was performed using real-time PCR. Dietary data was collected through questionnaires and analyzed using the Nutritionist Pro™ software. Quantification of cytokines was conducted using Bio-Plex Pro™ Human cytokine. The Astrand-Ryhming test was used to estimate the maximum oxygen volume and design the moderate-intensity structured exercise program ~ 75% heart rate (HR) RESULTS: After the intervention, both study groups significantly improved body composition (decreased weight, fat mass, waist circumference and abdominal obesity, p < 0.05). Besides, the diet-exercise group significantly decreased ASC mRNA expression, MCP-1, and MIP-1ß inflammatory cytokines compared to the diet group (p < 0.05). While in the diet group, MCP-1 and IL-8 exhibited significantly decreased levels (p < 0.05). In the diet-exercise group, a positive correlation between the atherogenic index and waist circumference was found (r = 0.822, p = 0.011), and a negative correlation was observed between the delta of ASC mRNA expression and IL-10 levels at the end of the intervention (r = - 0.627, p = 0.019). CONCLUSION: Low-grade chronic inflammation was attenuated through individualized exercise prescription and our findings highlight the role of the ASC gene in the inflammation of obese adults. TRIAL REGISTRATION: ClinicalTrials.gov , number NCT04315376 . Registered 20 March 2020-retrospectively registered.

Mitochondrial bound hexokinase type I in normal and streptozotocin diabetic rat retina.

Diabetic retinopathy is thought to be trigger by glucose- induced oxidative stress which leads to an increase of the mitochondrial permeability through opening the permeability transition pore (MTP). In several cell types, hexokinases interact with the mitochondria regulating MTP opening, avoiding cytochrome c release. We studied HK I mitochondrial proportion in control and streptozotocin-induced diabetic rat retinas. In the normal retina, 50% of HK I was linked to mitochondria, proportion that did not change up to 60 days of diabetes. Mitochondria from normal and diabetic rat retinas showed a limited swelling, and similar cytochrome c levels. G-6-P and glycogen content increased 3-6-fold in diabetic rat retinas, while lactate content did not vary. Results suggest that mitochondrial bound HK produce G-6-P and drove it to glycogen synthesis, controlling ROS production and lactate toxicity.

Glycine receptor is differentially expressed in the rat retina at early stages of streptozotocin-induced diabetes.

Diabetes mellitus is a metabolic disease that leads to several complications which include retinopathy. Neuronal abnormalities have been reported to appear before microvasculature alterations. We analyzed the expression levels of GlyR subunits in the retina at 7, 20, and 45 days after streptozotocin-induced diabetes to gain insight into the pathogenesis of diabetic retinopathy. We determined the mRNA and protein expression by qPCR and western blot, respectively. The mRNA and protein expression of the α1 subunit was not altered over the study period; however, they were slightly reduced in α2 yet statistically significant. While protein expression of α3 subunit was only reduced at 45 days diabetes. The mRNA and protein expression of the α4 subunit was remarkably decreased since day 7 of diabetes, remaining only ∼20% on day 45 of diabetes. Surprisingly, the mRNA of the ß subunit was highly increased, while its protein levels were not changed. The decrease in GlyR α subunits expression in the retina from diabetic animals suggest a perturbation in the inhibitory glycine signaling pathway, which might be related to the visual alterations observed in diabetes.

Recurrent moderate hypoglycemia exacerbates oxidative damage and neuronal death leading to cognitive dysfunction after the hypoglycemic coma.

Moderate recurrent hypoglycemia (RH) is frequent in Type 1 diabetes mellitus (TIDM) patients who are under intensive insulin therapy increasing the risk for severe hypoglycemia (SH). The consequences of RH are not well understood and its repercussions on neuronal damage and cognitive function after a subsequent episode of SH have been poorly investigated. In the current study, we have addressed this question and observed that previous RH during seven consecutive days exacerbated oxidative damage and neuronal death induced by a subsequent episode of SH accompanied by a short period of coma, in the parietal cortex, the striatum and mainly in the hippocampus. These changes correlated with a severe decrease in reduced glutathione content (GSH), and a significant spatial and contextual memory deficit. Administration of the antioxidant, N-acetyl-L-cysteine, (NAC) reduced neuronal death and prevented cognitive impairment. These results demonstrate that previous RH enhances brain vulnerability to acute hypoglycemia and suggests that this effect is mediated by the decline in the antioxidant defense and oxidative damage. The present results highlight the importance of an adequate control of moderate hypoglycemic episodes in TIDM.

Reversals and limitations on high-intensity, life-sustaining treatments.

IMPORTANCE: Critically ill patients often receive high-intensity life sustaining treatments (LST) in the intensive care unit (ICU), although they can be ineffective and eventually undesired. Determining the risk factors associated with reversals in LST goals can improve patient and provider appreciation for the natural history and epidemiology of critical care and inform decision making around the (continued) use of LSTs. METHODS: This is a single institution retrospective cohort study of patients receiving life sustaining treatment in an academic tertiary hospital from 2009 to 2013. Deidentified patient electronic medical record data was collected via the clinical data warehouse to study the outcomes of treatment limiting Comfort Care and do-not-resuscitate (DNR) orders. Extended multivariable Cox regression models were used to estimate the association of patient and clinical factors with subsequent treatment limiting orders. RESULTS: 10,157 patients received life-sustaining treatment while initially Full Code (allowing all resuscitative measures). Of these, 770 (8.0%) transitioned to Comfort Care (with discontinuation of any life-sustaining treatments) while 1,669 (16%) patients received new DNR orders that reflect preferences to limit further life-sustaining treatment options. Patients who were older (Hazard Ratio(HR) 1.37 [95% CI 1.28-1.47] per decade), with cerebrovascular disease (HR 2.18 [95% CI 1.69-2.81]), treated by the Medical ICU (HR 1.92 [95% CI 1.49-2.49]) and Hematology-Oncology (HR 1.87 [95% CI 1.27-2.74]) services, receiving vasoactive infusions (HR 1.76 [95% CI 1.28, 2.43]) or continuous renal replacement (HR 1.83 [95% CI 1.34, 2.48]) were more likely to transition to Comfort Care. Any new DNR orders were more likely for patients who were older (HR 1.43 [95% CI 1.38-1.48] per decade), female (HR 1.30 [95% CI 1.17-1.44]), with cerebrovascular disease (HR 1.45 [95% CI 1.25-1.67]) or metastatic solid cancers (HR 1.92 [95% CI 1.48-2.49]), or treated by Medical ICU (HR 1.63 [95% CI 1.42-1.86]), Hematology-Oncology (HR 1.63 [95% CI 1.33-1.98]) and Cardiac Care Unit-Heart Failure (HR 1.41 [95% CI 1.15-1.72]). CONCLUSION: Decisions to reverse or limit treatment goals occurs after more than 1 in 13 trials of LST, and is associated with older female patients, receiving non-ventilator forms of LST, cerebrovascular disease, and treatment by certain medical specialty services.

The exceptional value of intact forest ecosystems.

As the terrestrial human footprint continues to expand, the amount of native forest that is free from significant damaging human activities is in precipitous decline. There is emerging evidence that the remaining intact forest supports an exceptional confluence of globally significant environmental values relative to degraded forests, including imperilled biodiversity, carbon sequestration and storage, water provision, indigenous culture and the maintenance of human health. Here we argue that maintaining and, where possible, restoring the integrity of dwindling intact forests is an urgent priority for current global efforts to halt the ongoing biodiversity crisis, slow rapid climate change and achieve sustainability goals. Retaining the integrity of intact forest ecosystems should be a central component of proactive global and national environmental strategies, alongside current efforts aimed at halting deforestation and promoting reforestation.

Nitrosative Stress in the Rat Retina at the Onset of Streptozotocin-Induced Diabetes.

BACKGROUND/AIMS: Nitric oxide is a multifunctional molecule that can modify proteins via nitrosylation; it can also initiate signaling cascades through the activation of soluble guanylate cyclase. Diabetic retinopathy is the leading cause of blindness, but its pathogenesis is unknown. Multiple mechanisms including oxidative-nitrosative stress have been implicated. Our main goal was to find significant changes in nitric oxide (NO) levels and determine their association with nitrosative stress in the rat retina at the onset of diabetes. METHODS: Diabetes was induced by a single intraperitoneal administration of streptozotocin. The possible nitric oxide effects on the rat retina were evaluated by the presence of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a specific marker for NO-producing neurons, detected by histochemistry performed on whole retinas and retina sections. Immunohistochemistry was also performed on retina sections for iNOS, 3-nitrotyrosine (3-NT) and glial fibrillary acidic protein (GFAP). Retinal nitric oxide levels were assessed by measuring total nitrate/nitrite concentrations. Retinal cGMP levels were determined by radioimmunoassay. Western blots for nitrotyrosine (3-NT) and oxidized proteins were performed. RESULTS: In the present study, we found increased activity of NADPH-diaphorase and iNOS immunoreactivity in the rat retina at the onset of diabetes; this increase correlated with a remarkable increase in NO levels as early as 7 days after the onset of diabetes. However, cGMP levels were not modified by diabetes, suggesting that NO did not activate its signaling cascade. Even so, Western blots revealed a progressive increase in nitrated proteins at 7 days after diabetes induction. Likewise, positive nitrotyrosine immunolabeling was observed in the photoreceptor layer, ganglion cell layer, inner nuclear layer and some Müller cell processes in the retinas of diabetic rats. In addition, levels of oxidized proteins were increased in the retina early after diabetes induction; these levels were reduced by the administration of L-NAME. In addition, stress in Müller cells was determined by immunoreactivity to the glial fibrillary acidic protein. CONCLUSIONS: Our findings indicated the occurrence of nitrosative stress at the onset of diabetes in the rat retina and emphasized the role of NO in retinal function and the pathogenesis of retinopathy.

Glycine receptor subunits expression in the developing rat retina.

BACKGROUND AND METHODS: Glycine receptor (GlyR) consists of two α (1-4) and three ß subunits. Considerable evidence indicates that the adult retina expresses the four types of α subunits; however, the proportion of these subunits in adult and immature retina is almost unknown. In this report we have studied mRNA and the protein expression of GlyR subunits in the retina during postnatal rat development by Real-Time qRT-PCR and western blot. RESULTS: mRNA and protein expression indicated a gradual increase of the α1, α3, α4 and ß GlyR subunits during postnatal ages tested. The mRNA ß subunit showed higher expression levels (∼3 fold) than those observed for the α1 and α3 subunits. Very interestingly, the α2 GlyR subunit had the highest expression in the retina, even in the adult. CONCLUSIONS: These results revealed the expression of GlyR at early postnatal ages, supporting its role in retina development. In addition, our results indicated that the adult retina expressed a high proportion of the α2 subunit, suggesting the expression of monomeric and/or heteromeric receptors. A variety of studies are needed to further characterize the role of the specific subunits in both adult and immature retina.

Potential Role of Endoplasmic Reticulum Stress in Pathogenesis of Diabetic Retinopathy.

Diabetes mellitus is a metabolic disease that leads to several complications which include retinopathy. Multiple biochemical abnormalities have been proposed to explain the development of retinopathy, including oxidative stress. Although the existence of oxidative stress has been established in the retina from long standing diabetic animals, pathogenesis and progression of retinopathy remain unclear. In order to gain insight into the pathogenesis of diabetic retinopathy, we analyzed the levels of different oxidative stress biomarkers in the retina at early stages during the progress of streptozotocin-induced diabetes. No significant changes in glutathione content, expression of NADPH-oxidase, levels of lipid peroxidation, nor production of free radicals were observed in the retina up to 45 days of diabetes induction. Likewise, a transient decrease in aconitase activity, parallel to an increase in the superoxide dismutase activity was observed at 20 days of hyperglycemia, suggesting a high capacity of retina to maintain its redox homeostasis, at least at early stages of diabetes. Nonetheless, we found an early and time-dependent increase in the levels of oxidized proteins, which was not affected by the administration of the antioxidant quercetin. Also, positive immunoreactivity to the reticulum stress protein CHOP was found in glial Müller cells of diabetic rat retinas. These findings suggest the occurrence of endoplasmic reticulum stress as a primary event in retina pathogenesis in diabetes.

Control of glycogen content in retina: allosteric regulation of glycogen synthase.

Retinal tissue is exceptional because it shows a high level of energy metabolism. Glycogen content represents the only energy reserve in retina, but its levels are limited. Therefore, elucidation of the mechanisms controlling glycogen content in retina will allow us to understand retina response under local energy demands that can occur under normal and pathological conditions. Thus, we studied retina glycogen levels under different experimental conditions and correlated them with glucose-6-phosphate (G-6-P) content and glycogen synthase (GS) activity. Glycogen and G-6-P content were studied in ex vivo retinas from normal, fasted, streptozotocin-treated, and insulin-induced hypoglycemic rats. Expression levels of GS and its phosphorylated form were also analyzed. Ex vivo retina from normal rats showed low G-6-P (14±2 pmol/mg protein) and glycogen levels (43±3 nmol glycosyl residues/mg protein), which were increased 6 and 3 times, respectively, in streptozotocin diabetic rats. While no changes in phosphorylated GS levels were observed in any condition tested, a positive correlation was found between G-6-P levels with GS activity and glycogen content. The results indicated that in vivo, retina glycogen may act as an immediately accessible energy reserve and that its content was controlled primarily by G-6-P allosteric activation of GS. Therefore, under hypoglycemic situations retina energy supply is strongly compromised and could lead to the alterations observed in type 1 diabetes.

Insulin stimulated-glucose transporter Glut 4 is expressed in the retina.

The vertebrate retina is a very metabolically active tissue whose energy demands are normally met through the uptake of glucose and oxygen. Glucose metabolism in this tissue relies upon adequate glucose delivery from the systemic circulation. Therefore, glucose transport depends on the expression of glucose transporters. Here, we show retinal expression of the Glut 4 glucose transporter in frog and rat retinas. Immunohistochemistry and in situ hybridization studies showed Glut 4 expression in the three nuclear layers of the retina: the photoreceptor, inner nuclear and ganglionar cell layers. In the rat retina immunoprecipitation and Western blot analysis revealed a protein with an apparent molecular mass of 45 kDa. ¹4C-glucose accumulation by isolated rat retinas was significantly enhanced by physiological concentrations of insulin, an effect blocked by inhibitors of phosphatidyl-inositol 3-kinase (PI3K), a key enzyme in the insulin-signaling pathway in other tissues. Also, we observed an increase in ³H-cytochalasin binding sites in the presence of insulin, suggesting an increase in transporter recruitment at the cell surface. Besides, insulin induced phosphorylation of Akt, an effect also blocked by PI3K inhibition. Expression of Glut 4 was not modified in retinas of a type 1 diabetic rat model. To our knowledge, our results provide the first evidence of Glut4 expression in the retina, suggesting it as an insulin- responsive tissue.

Levels of neural progenitors in the hippocampus predict memory impairment and relapse to drug seeking as a function of excessive methamphetamine self-administration.

Methamphetamine affects the hippocampus, a brain region crucial for learning and memory, as well as relapse to drug seeking. Rats self-administered methamphetamine for 1 h twice weekly (intermittent-short-I-ShA), 1 h daily (limited-short-ShA), or 6 h daily (extended-long-LgA) for 22 sessions. After 22 sessions, rats from each access group were withdrawn from self-administration and underwent spatial memory (Y-maze) and working memory (T-maze) tests followed by extinction and reinstatement to methamphetamine seeking or received one intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) to label progenitors in the hippocampal subgranular zone (SGZ) during the synthesis phase. Two-hour-old and 28-day-old surviving BrdU-immunoreactive cells were quantified. I-ShA rats performed better on the Y-maze and had a greater number of 2-h-old SGZ BrdU cells than nondrug controls. LgA rats, but not ShA rats, performed worse on the Y- and T-maze and had a fewer number of 2-h-old SGZ BrdU cells than nondrug and I-ShA rats, suggesting that new hippocampal progenitors, decreased by methamphetamine, were correlated with impairment in the acquisition of new spatial cues. Analyses of addiction-related behaviors after withdrawal and extinction training revealed methamphetamine-primed reinstatement of methamphetamine-seeking behavior in all three groups (I-ShA, ShA, and LgA), and this effect was enhanced in LgA rats compared with I-ShA and ShA rats. Protracted withdrawal from self-administration enhanced the survival of SGZ BrdU cells, and methamphetamine seeking during protracted withdrawal enhanced Fos expression in the dentate gyrus and medial prefrontal cortex in LgA rats to a greater extent than in ShA and I-ShA rats. These results indicate that changes in the levels of the proliferation and survival of hippocampal neural progenitors and neuronal activation of hippocampal granule cells predict the effects of methamphetamine self-administration (limited vs extended access) on cognitive performance and relapse to drug seeking and may contribute to the impairments that perpetuate the addiction cycle.