Repairing brain after stroke: a review on post-ischemic neurogenesis

Stroke is devastating as currently no therapies are available that can prevent stroke-induced neurological dysfunction in humans. With the recent observations that acute insults to adult brain stimulate new neuronal formation in various species of animals, optimism is building for a possible regeneration of stroke-damaged brain. This article reviewed the advances in the understanding of the molecular mechanisms of the various steps of neurogenesis with an emphasis on the endogenous mediators and exogenous promoters of neural progenitor proliferation, migration and survival in the post-ischemic adult brain.     

Permeability of the post-ischemic rat aorta.

The time course of the vascular permeability disorder following upon acute hypoxia and the fate of plasma substances entering the vessel wall in consequence of increased permeability were studied on the rat abdominal aorta rendered hypoxic for one hour by double ligature and recirculated for periods ranging from one hour to 30 days. The distribution and quantity of mural plasma inhibition were determined histochemically by means of a colloidal iron tracer and Prussian blue reaction and by photometric analysis, respectively. Plasma imbibition reached its maximum after recirculation for 24 to 48 hours and fell to an almost normal level after 10 days. Administration of the colloidal iron tracer on the second day of recirculation, when the permeability disorder was at its peak, showed plasma imbibition in every layer of the vessel wall. At seven days it was restricted to the outer third of the media and the adventitia. The endothelium is acting as the main barrier to mural plasma imbibition and in the case of a permeability disorder only the elastic lamellae constitute a temporary mechanical obstacle to the ingression of plasma.     

Gender and post-ischemic recovery of hypertrophied rat hearts

Background

Cardiac mortality in Duchenne muscular dystrophy (DMD) has recently become important, because risk of respiratory failure has been reduced due to widespread use of the respirator. The cardiac involvement is characterized by distinctive electrocardiographic abnormalities or dilated cardiomyopathy, but the pathogenesis has remained obscure. In research on DMD, Golden retriever-based muscular dystrophy (GRMD) has attracted much attention as an animal model because it resembles DMD, but GRMD is very difficult to maintain because of their severe phenotypes. We therefore established a line of dogs with Beagle-based canine X-linked muscular dystrophy in Japan (CXMD_J) and examined the cardiac involvement.

Methods

The cardiac phenotypes of eight CXMD_J and four normal male dogs 2 to 21 months of age were evaluated using electrocardiography, echocardiography, and histopathological examinations.

Results

Increases in the heart rate and decreases in PQ interval compared to a normal littermate were detected in two littermate CXMD_J dogs at 15 months of age or older. Distinct deep Q-waves and increase in Q/R ratios in leads II, III, and aVF were detected by 6–7 months of age in all CXMD_J dogs. In the echocardiogram, one of eight of CXMD_J dogs showed a hyperechoic lesion in the left ventricular posterior wall at 5 months of age, but the rest had not by 6–7 months of age. The left ventricular function in the echocardiogram indicated no abnormality in all CXMD_J dogs by 6–7 months of age. Histopathology revealed myocardial fibrosis, especially in the left ventricular posterobasal wall, in three of eight CXMD_J dogs by 21 months of age.

Conclusion

Cardiac involvement in CXMD_J dogs is milder and has slower progression than that described in GRMD dogs. The distinct deep Q-waves have been ascribed to myocardial fibrosis in the posterobasal region of the left ventricle, but our data showed that they precede the lesion on echocardiogram and histopathology. These findings imply that studies of CXMD_J may reveal not only another causative mechanism of the deep Q-waves but also more information on the pathogenesis in the dystrophin-deficient heart.     

Local ASIC3 modulates pain and disease progression in a rat model of osteoarthritis

Background

Recent data have suggested a relationship between acute arthritic pain and acid sensing ion channel 3 (ASIC3) on primary afferent fibers innervating joints. The purpose of this study was to clarify the role of ASIC3 in a rat model of osteoarthritis (OA) which is considered a degenerative rather than an inflammatory disease.

Methods

We induced OA via intra-articular mono-iodoacetate (MIA) injection, and evaluated pain-related behaviors including weight bearing measured with an incapacitance tester and paw withdrawal threshold in a von Frey hair test, histology of affected knee joint, and immunohistochemistry of knee joint afferents. We also assessed the effect of ASIC3 selective peptide blocker (APETx2) on pain behavior, disease progression, and ASIC3 expression in knee joint afferents.

Results

OA rats showed not only weight-bearing pain but also mechanical hyperalgesia outside the knee joint (secondary hyperalgesia). ASIC3 expression in knee joint afferents was significantly upregulated approximately twofold at Day 14. Continuous intra-articular injections of APETx2 inhibited weight distribution asymmetry and secondary hyperalgesia by attenuating ASIC3 upregulation in knee joint afferents. Histology of ipsilateral knee joint showed APETx2 worked chondroprotectively if administered in the early, but not late phase.

Conclusions

Local ASIC3 immunoreactive nerve is strongly associated with weight-bearing pain and secondary hyperalgesia in MIA-induced OA model. APETx2 inhibited ASIC3 upregulation in knee joint afferents regardless of the time-point of administration. Furthermore, early administration of APETx2 prevented cartilage damage. APETx2 is a novel, promising drug for OA by relieving pain and inhibiting disease progression.     

Importance of urinary enzymes as a marker of post-ischemic proximal tubular involvement in rat

We have studied in rats the influence of renal ischemia on urinary excretion of three brush border membrane enzymes (gamma glutamyl transferase, alkaline phosphatase and leucine aminopeptidase) and of a lysosomal one (N-acetyl-B-D-glucosaminidase). Urines were collected over 24 hours periods during three days before and after a 45 minutes renal artery clamping. Urinary GGT, PAL and LAP excretion were significantly increased on the first day after renal ischemia, but returns to normal values on the second day. Urinary NAG activity increases on the first day, but contrary to the latter enzymes, reached to normal values only on the third day. Enzymuria seems to be a useful marker of tubular injury occurring after a temporary renal ischemia in the rat.     

Opioids modulate periodicity rather than efficacy of peristaltic waves in the guinea pig ileum in vitro

The influence of opioid receptor blockade by naloxone and opioid receptor activation by opioids on peristalsis was studied in isolated segments of the guinea pig ileum.1. (-)Naloxone, but not (+)naloxone, increased the mean number of peristaltic waves per min within periods of elevated intraluminal pressure. Naloxone tended to modify intermittent peristalsis into ongoing peristalsis, whereas opioids worked in an opposite fashion. 2. Maximum amplitudes of luminal volume displacement during single peristaltic waves were not decreased by opioids. (-)Naloxone, however, applied to non-pretreated segments, decreased transitorily the efficacy of single peristaltic waves to a small, but statistically significant degree 3. Enhancement of peristalsis by naloxone decreased over time, although enough naloxone was present to occupy all opioid receptors. This suggests that opioid receptor blockade induces some compensatory mechanism.     

Black tea polyphenols modulate xenobiotic-metabolizing enzymes, oxidative stress and adduct formation in a rat hepatocarcinogenesis model

The present study was designed to investigate the modulatory effects of black tea polyphenols (Polyphenon-B) on phase I and phase II xenobiotic-metabolizing enzymes and oxidative stress in a rat model of hepatocellular carcinoma (HCC). Liver tumours induced in male Sprague-Dawley rats by dietary administration of rho-dimethylaminoazobenzene (DAB) increased cytochrome P450 (total and CYP1A1, 1A2 and 2B isoforms), cytochrome b(5), cytochrome b(5) reductase, glutathione S-transferase (GST total and GST-P isoform) and gamma-glutamyltranspeptidase (GGT) with decrease in quinone reductase (QR). This was accompanied by enhanced lipid and protein oxidation and compromised antioxidant defences associated with increased expression of the oxidative stress markers 4-hydroxynonenal (4-HNE), anti-hexanoyl lysine (HEL), dibromotyrosine (DiBrY) and 8-hydroxy 2-deoxyguanosine (8-OHdG). Dietary administration of Polyphenon-B effectively suppressed DAB-induced hepatocarcinogenesis, as evidenced by reduced preneoplastic and neoplastic lesions, modulation of xenobiotic-metabolizing enzymes and amelioration of oxidative stress. Thus, it can be concluded that Polyphenon-B acts as an effective chemopreventive agent by modulating xenobiotic-metabolizing enzymes and mitigating oxidative stress in an in vivo model of hepatocarcinogenesis.     

Black tea polyphenols modulate xenobiotic-metabolizing enzymes,oxidative stress and adduct formation in a rat hepatocarcinogenesis model

The present study was designed to investigate the modulatory effects of black tea polyphenols (Polyphenon-B) on phase I and phase II xenobiotic-metabolizing enzymes and oxidative stress in a rat model of hepatocellular carcinoma (HCC). Liver tumours induced in male Sprague-Dawley rats by dietary administration of ρ-dimethylaminoazobenzene (DAB) increased cytochrome P450 (total and CYP1A1, 1A2 and 2B isoforms), cytochrome b₅, cytochrome b₅ reductase, glutathione S-transferase (GST total and GST-P isoform) and gamma-glutamyltranspeptidase (GGT) with decrease in quinone reductase (QR). This was accompanied by enhanced lipid and protein oxidation and compromised antioxidant defences associated with increased expression of the oxidative stress markers 4-hydroxynonenal (4-HNE), anti-hexanoyl lysine (HEL), dibromotyrosine (DiBrY) and 8-hydroxy 2-deoxyguanosine (8-OHdG). Dietary administration of Polyphenon-B effectively suppressed DAB-induced hepatocarcinogenesis, as evidenced by reduced preneoplastic and neoplastic lesions, modulation of xenobiotic-metabolizing enzymes and amelioration of oxidative stress. Thus, it can be concluded that Polyphenon-B acts as an effective chemopreventive agent by modulating xenobiotic-metabolizing enzymes and mitigating oxidative stress in an in vivo model of hepatocarcinogenesis.     

Effects of intranasal guanosine administration on brain function in a rat model of ischemic stroke

Ischemic stroke is a major cause of morbidity and mortality worldwide and only few affected patients are able to receive treatment, especially in developing countries. Detailed pathophysiology of brain ischemia has been extensively studied in order to discover new treatments with a broad therapeutic window and that are accessible to patients worldwide. The nucleoside guanosine (Guo) has been shown to have neuroprotective effects in animal models of brain diseases, including ischemic stroke. In a rat model of focal permanent ischemia, systemic administration of Guo was effective only when administered immediately after stroke induction. In contrast, intranasal administration of Guo (In-Guo) was effective even when the first administration was 3 h after stroke induction. In order to validate the neuroprotective effect in this larger time window and to investigate In-Guo neuroprotection under global brain dysfunction induced by ischemia, we used the model of thermocoagulation of pial vessels in Wistar rats. In our study, we have found that In-Guo administered 3 h after stroke was capable of preventing ischemia-induced dysfunction, such as bilateral suppression and synchronicity of brain oscillations and ipsilateral cell death signaling, and increased permeability of the blood-brain barrier. In addition, In-Guo had a long-lasting effect on preventing ischemia-induced motor impairment. Our data reinforce In-Guo administration as a potential new treatment for brain ischemia with a more suitable therapeutic window.

     

Lipid nanocapsules loaded with rhenium-188 reduce tumor progression in a rat hepatocellular carcinoma model

Background

Due to their nanometric scale (50 nm) along with their biomimetic properties, lipid nanocapsules loaded with Rhenium-188 (LNC¹⁸⁸Re-SSS) constitute a promising radiopharmaceutical carrier for hepatocellular carcinoma treatment as its size may improve tumor penetration in comparison with microspheres devices. This study was conducted to confirm the feasibility and to assess the efficacy of internal radiation with LNC¹⁸⁸Re-SSS in a chemically induced hepatocellular carcinoma rat model.

Methodology/Principal Findings

Animals were treated with an injection of LNC¹⁸⁸Re-SSS (80 MBq or 120 MBq). The treated animals (80 MBq, n = 12; 120 MBq, n = 11) were compared with sham (n = 12), blank LNC (n = 7) and ¹⁸⁸Re-perrhenate (n = 4) animals. The evaluation criteria included rat survival, tumor volume assessment, and vascular endothelial growth factor quantification. Following treatment with LNC¹⁸⁸Re-SSS (80 MBq) therapeutic efficiency was demonstrated by an increase in the median survival from 54 to 107% compared with control groups with up to 7 long-term survivors in the LNC¹⁸⁸Re-SSS group. Decreased vascular endothelial growth factor expression in the treated rats could indicate alterations in the angiogenesis process.

Conclusions/Significance

Overall, these results demonstrate that internal radiation with LNC¹⁸⁸Re-SSS is a promising new strategy for hepatocellular carcinoma treatment.     

ppGpp and polyphosphate modulate cell cycle progression in Caulobacter crescentus

Caulobacter crescentus differentiates from a motile, foraging swarmer cell into a sessile, replication-competent stalked cell during its cell cycle. This developmental transition is inhibited by nutrient deprivation to favor the motile swarmer state. We identify two cell cycle regulatory signals, ppGpp and polyphosphate (polyP), that inhibit the swarmer-to-stalked transition in both complex and glucose-exhausted media, thereby increasing the proportion of swarmer cells in mixed culture. Upon depletion of available carbon, swarmer cells lacking the ability to synthesize ppGpp or polyP improperly initiate chromosome replication, proteolyze the replication inhibitor CtrA, localize the cell fate determinant DivJ, and develop polar stalks. Furthermore, we show that swarmer cells produce more ppGpp than stalked cells upon starvation. These results provide evidence that ppGpp and polyP are cell-type-specific developmental regulators.     

Brain proteomics identifies potential simvastatin targets in acute phase of stroke in a rat embolic model

Finding an efficient neuroprotectant is of urgent need in the field of stroke research. The goal of this study was to test the effect of acute simvastatin administration after stroke in a rat embolic model and to explore its mechanism of action through brain proteomics. To that end, male Wistar rats were subjected to a Middle Cerebral Arteria Occlusion and simvastatin (20 mg/kg s.c) (n = 11) or vehicle (n = 9) were administered 15 min after. To evaluate the neuroprotective mechanisms of simvastatin, brain homogenates after 48 h were analyzed by two‐dimensional fluorescence Difference in Gel Electrophoresis (DIGE) technology. We confirmed that simvastatin reduced the infarct volume and improved neurological impairment at 48 h after the stroke in this model. Considering our proteomics analysis, 66 spots, which revealed significant differences between groups, were analyzed by matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry allowing the identification of 27 proteins. From these results, we suggest that simvastatin protective effect can be partly explained by the attenuation of the oxidative and stress response at blood–brain barrier level after cerebral ischemia. Interestingly, analyzing one of the proteins (HSP75) in plasma from stroke patients who had received simvastatin during the acute phase, we confirmed the results found in the pre‐clinical model.

     

Factors affecting the post-ischemic recuperation of isolated perfused rat heart

A number of cardioplegic solutions have been described for the reduction of cellular damage during ischemic cardiac arrest. Using an isolated working rat heart model, we have attempted to precise some of the factors affecting the post-ischemic recovery of myocardial tissue after a 30-min period of total ischemia at 37 degrees C. The results indicate that procaine (1 mM) is able to afford some protective against normothermic ischemia while this protective effect remains consistently lower than that of the St. Thomas' Hospital solution (procaine + high K+ + high Mg2+; JYNGE et al., 1977). On the other hand, hearts from rats of the Wistar strain consistently exhibit a significantly better degree of recovery than do hearts from rats of the Shermann strain. When hearts were perfused at different levels of preload (1 or 2 kPa) and afterload (8 or 10 kPa), post-ischemic recovery was better in hearts with lower levels of cardiac work. Glucose, insulin and DL-propranolol which have been shown to exert a protective effect in isolated rat hearts with regional ischemia failed to protect the heart in the present experimental conditions. No clear correlation does exist between the post-ischemic recovery and the enzymatic assessment of myocardial cell damage.     

The skeletal muscle Wnt pathway may modulate insulin resistance and muscle development in a diet-induced obese rat model

The Wnt signaling pathway is involved in lipid metabolism and obesity development. Skeletal muscle, a pivotal tissue for metabolism, is regulated by the Wnt signaling. However, little is known of this pathway's involvement in insulin sensitivity and myogenesis in animals. The current study focused on the potential role of Wnt signaling in insulin sensitivity and myogenic events and its further impact on intramuscular fat accumulation. Obesity resistant (OR) and obesity prone (OP) rats were fed a high-fat (HF, 45% kcal fat) diet for 13 weeks. Body weight and circulating triglyceride (TG) were measured and gastrocnemius muscle was collected for analysis of gene expression and protein amount. OP rats had higher body weight and blood TG than OR, and our study demonstrated that the skeletal muscle of OR and OP rats had different levels of β-catenin, which also corresponded to the expression of Wnt downstream genes. The expression of insulin receptor substrate (IRS) was significantly lower in OP than OR skeletal muscle, as was the protein amount of phosphorylated Akt, myocyte enhancer factor-2 (MEF2), and GLUT4. Expression of Myogenic regulatory factor (Myf) 5 and Myf3 (MyoD) were decreased significantly in OP skeletal muscle when compared to OR. Additionally, intramuscular fat was higher in OP than in OR rats. Thus, we propose that the differential Wnt signaling in the skeletal muscle of OR and OP rats is highly likely associated with the differences in insulin sensitivity and myogenic capability in these two strains.     

Induction of cytokine expression in rat post-ischemic sinoatrial node (SAN)

The aim of this study was to determine the spatial and temporal expression of various pro-inflammatory cytokines in the peri-sinoatrial nodal area after atrial infarction. Rats were subjected to permanent atrial infarction, in particular, sinoatrial node (SAN) infarction and sacrificed at various time points up to 7 days. Real-time polymerase chain reaction analysis demonstrated that mRNA levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta, interleukin-6, and transforming growth factor beta 1 (TGF-beta(1)) were upregulated in the peri-sinoatrial nodal area after atrial infarction. Immunostaining for TNF-alpha and TGF-beta(1) proteins revealed that both cytokines were expressed persistently up to 7 days after atrial infarction around the peri-sinoatrial nodal area. Furthermore, the infiltrating inflammatory cells immunoreactive for both cytokines were predominant within the infarct SAN. In situ hybridization analysis showed that TNF-alpha gene expression was enhanced in the inflammatory cells and myocardium within the peri-sinoatrial nodal area in response to the infarction. These results provide evidence for the local expression of cytokines in the post-ischemic peri-sinoatrial nodal area, suggesting that the upregulation of the cytokines might be associated with the atrial arrhythmia observed after acute myocardial infarction.     

Diacylglycerols modulate insulin action in rat adipocytes

Effect of 1,2-diacylglycerols on the insulin receptor function and insulin action in rat adipocytes was studied. 1,2-dioctanoylglycerol (100 micrograms/ml) did not alter insulin binding but it did stimulate phosphorylation of the beta-subunit of the insulin receptor as well as its tyrosine kinase activity. However, dioctanoylglycerol inhibited insulin-stimulated receptor autophosphorylation. This concentration of dioctanoylglycerol inhibited insulin-stimulated CO2 metabolism, lipogenesis and 3-O-methyl-glucose transport in a dose-dependent manner but did not alter any of these bioeffects in absence of insulin. While there was no direct link between diacylglycerol effect on tyrosine kinase activity of the insulin receptor and insulin action in rat adipocytes, the parallel inhibition of insulin-stimulated receptor autophosphorylation and insulin bioeffects by dioctanoylglycerol suggests its direct or indirect role in insulin signalling in rat fat cells.