Optimising the Photothrombotic Model of Stroke in the C57BI/6 and FVB/N Strains of Mouse

The photothrombotic stroke model relies on the interaction between photosensitive-dye and light for clot formation. Interestingly, the relationship between the length of light exposure and stroke-outcome has never been examined. This model has yet to be established in the FVB/N strain, even though stroke-outcomes are strain-specific. Therefore, this study aimed to examine the effect of different lengths of light exposure in two strains of mice on photothrombotic stroke. Male FVB/N and C57Bl/6 mice were subjected to stroke using 15, 18, or 20-mins light exposure. Mice underwent functional testing for up to 7 days. Infarct volume was assessed with thionin staining, and cellular responses to injury analysed via immunofluorescence. Blood brain barrier (BBB) breakdown was assessed using Evans blue dye. Increasing light exposure from 15 to 20-minutes increased infarct volume but not functional deficit. Interestingly, there were strain-specific differences in functional outcomes, with FVB/N mice having less deficit on the hanging wire test than C57BI/6 after 15-minutes of light exposure. The opposite was seen in the adhesive removal test. There was no difference in number of neurons, astrocytes, microglia, macrophages, and T cells between the strains, despite FVB/N mice demonstrating greater BBB breakdown and an enlarged spleen post-stroke. Increasing light exposure systematically increases infarct volume but does not worsen functional outcomes. FVB/N and C57Bl/6 mice exhibit subtle differences in functional outcomes post stroke, which highlights the need to choose tests which are appropriate for the mouse strain being used.

Residual Adhesive Removal Methods for Rebonding of Debonded Orthodontic Metal Brackets: Systematic Review and Meta-Analysis

Debonding of orthodontic brackets is a common occurrence during orthodontic treatment. Therefore, the best option for treating debonded brackets should be indicated. This study aimed to evaluate the bond strength of rebonded brackets after different residual adhesive removal methods. This systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. PubMed, Web of Science, The Cochrane Library, SciELO, Scopus, LILACS, IBECS, and BVS databases were screened up to December 2020. Bond strength comparisons were made considering the method used for removing the residual adhesive on the bracket base. A total of 12 studies were included for the meta-analysis. Four different adhesive removal methods were identified: sandblasting, laser, mechanical grinding, and direct flame. When compared with new orthodontic metallic brackets, bond strength of debonded brackets after air abrasion (p = 0.006), mechanical grinding (p = 0.007), and direct flame (p < 0.001) was significantly lower. The use of an erbium-doped yttrium aluminum garnet (Er:YAG) laser showed similar shear bond strength (SBS) values when compared with those of new orthodontic brackets (p = 0.71). The Er:YAG laser could be considered an optimal method for promoting the bond of debonded orthodontic brackets. Direct flame, mechanical grinding, or sandblasting are also suitable, obtaining clinically acceptable bond strength values.

Evaluation of Deinking Performance of Active Calcium Silicate Filled Paper

The deinking effect of printing paper filled with CaSiO3 and CaSiO3/French chalk was investigated. As a result, it shows that the two kinds of paper samples have better deinking effect, and the whiteness of the former is slightly higher than that of the latter. However, the strength index of the former is slightly lower than that of the latter. The adhesive removal effect of the paper sample filled with CaSiO3 is better than that of the paper filled with CaSiO3/ French chalk powder.

Morphological characteristic of intracerebral hemorrhage in rats and correlation of its volume with results of behavioral tests

The intracerebral hemorrhage is associated with severe complications and high mortality. Currently there are no effective methods of treatment of this disease while the standard collagenase model of intracerebral hemorrhage is not described sufficiently. Objective. To analyze morphological characteristics of the collagenase model of intracerebral hemorrhage, and develop the regression formula predicting the hemorrhage volume based on the results of behavioral tests. Materials and methods. The experiments were carried out on 7 white male rats weighing 250-400 g aged 11-13 months. All animals underwent surgery to simulate intracerebral hemorrhage. Rats were anesthetized and then, stereotactically, using a needle with a diameter of 0.47 mm, 0.2 units of collagenase type IV were slowly injected into the left striatum. The day after the intracerebral hemorrhage, functional disabilities developed in rats were studied using beam walking test, neurological score test and adhesive removal test. Immediately after performing behavioral tests, the rats were sacrificed by decapitation. After the brain formalin fixation, serial sections on a vibromicrotome of 200 µm thick each in the anteroposterior direction with following morphological examination were made. Results. It was revealed that the collagenase model of intracerebral hemorrhage is associated with a large variability of the hemorrhage volume. It also had an irregular rugged shape and marks of repeated diapedetic hemorrhages of about 0.6 mm depth. The center of intracerebral hemorrhage along the anteroposterior axis was in average 0.5 mm posterior of the actual site of collagenase injection.The combined use of the neurological score test, the beam walking test and the adhesive removal test in the collagenase model can help estimate the probable intracerebral hemorrhage volume on the 1st day using the regression formula. Conclusions. Technical details identified in our study can help researchers in planning and conduction of correct experiments related to intracerebral hemorrhage.

Differential Effects of Physical and Social Enriched Environment on Angiogenesis in Male Rats After Cerebral Ischemia/Reperfusion Injury

Different housing conditions, including housing space and the physiological and social environment, may affect rodent behavior. Here, we examined the effects of different housing conditions on post-stroke angiogenesis and functional recovery to clarify the ambiguity about environmental enrichment and its components. Male rats in the model groups underwent right middle cerebral artery occlusion (MCAO) followed by reperfusion. The MCAO rats were divided into four groups: the physical enrichment (PE) group, the social enrichment (SE) group, the combined physical and social enrichment (PSE) group and the ischemia/reperfusion + standard conditioning (IS) group. The rats in the sham surgery (SS) group were housed under standard conditions. In a set of behavioral tests, including the modified Neurological Severity Score (mNSS), rotarod test, and adhesive removal test, we demonstrated that the animals in the enriched condition groups exhibited significantly improved neurological functions compared to those in the standard housing group. Smaller infarction volumes were observed in the animals of the PSE group by MRI detection. The enriched conditions increased the microvessel density (MVD) in the ischemic boundary zone, as revealed by CD31 immunofluorescent staining. The immunochemical and q-PCR results further showed that environmental enrichment increased the expression levels of angiogenic factors after ischemia/reperfusion injury. Our data suggest that all three enrichment conditions promoted enhanced angiogenesis and functional recovery after ischemia/reperfusion injury compared to the standard housing, while only exposure to the combination of both physical and social enrichment yielded optimal benefits.

Abstract P795: Activating Transcription Factor 3 Improves Stroke Recovery Through Reduction of Neuronal Damage and Neuroinflammation

Background and Purpose: Activating transcription factor 3 (ATF3), a member of the ATF/CREB family, is upregulated in the early phase of various stresses including ischemic stroke. The role of ATF3 in ischemic stroke injury has not been fully elucidated. Hypothesis: ATF3 improves stroke outcomes through reduction of neuronal damage and neuroinflammation. Methods: Permanent distal middle cerebral artery occlusion (pMCAO) was performed in 8-week-old male and female C57BL/6J (WT) and ATF3 knockout (ATF3 -/- ) mice. The sensorimotor functions were analyzed 3 days after pMCAO through adhesive removal and corner tests. Infarct volumes and injured neurons were quantified on Nissl stained and Fluoro-Jade C (FJC) stained sections. The cell-specific expression of ATF3 was analyzed by co-staining ATF3 antibody with antibodies specific to NeuN (Neuron), CD68 (microglia/macrophage) and GFAP (astrocyte). The expression of ATF3 in injured neurons was analyzed by ATF3 and FJC double labeling. Results: ATF3 expression was detected exclusively in neurons in the infarct area 1 day after pMCAO. There were a few ATF3 + CD68 + microglia/macrophages at the peri-infarct regions 2 and 3 days after pMCAO. Almost all FJC + neurons were ATF3 positive. No ATF3 expression was detected in astrocytes. Compared to WT mice, ATF3 -/- mice took longer time to remove the adhesive from their right paws (p<0.001) in adhesive removal test and more left turns in corner test (p<0.001) 3 days after pMCAO. ATF3 -/- mice also had a larger infarct volume (p = 0.014), more FJC + neurons (p=0.002) and CD68 + microglia/macrophages (p=0.003) in the peri-infarct regions than WT mice. Conclusions: Deletion of ATF3 exacerbates neuronal injury, neuroinflammation, and sensorimotor dysfunction in stroke mice, suggesting that upregulation of ATF3 at early stage of stroke improves ischemic stroke recovery through reduction of neuronal damage and neuroinflammation.

Abstract P759: Microglial EZH2 Inhibition Leads to Neuroprotection After Stroke in Aged Mice

Introduction: Ischemic stroke results in activation of microglia, which may polarize towards a pro-inflammatory (M1) phenotype and/or an anti-inflammatory (M2) phenotype. Enhancer of zeste homolog (EZH) 2 is a histone-lysine N-methyltransferase enzyme. Accumulating evidence has suggested EZH2 is key modulator of microglia polarization by epigenetically regulating gene expression. We here investigated whether microglial-specific deletion of EZH2 leads to a beneficial protective effect in stroke in vivo . We further tested the therapeutical potential of an EZH2 inhibitor after stroke. Methods: Aged male mice were subjected to 60-minutes middle cerebral artery stroke. Tamoxifen administration was started 30 days prior to stroke to induce genetic deletion of microglial EZH2 in CX3CR1-creER/EZH2-floxed mice. EZH2 floxed mice were used as controls. EZH2 inhibitor GSK343 was I.P. injected (once a day for two consecutive days), starting three hours after stroke onset. Mice were sacrificed for immunohistochemistry and crystal violet staining (brain infarct assay) after behavior tests at 3 days after stroke. Results: The expression of microglial EZH2 was significantly abrogated in KO mice compared to the control floxed mice (144±15.43 vs. 50.65±4.99 cells/mm 2 , N=5/each group, P<0.01). EZH2 deletion reduced brain infarct volume (29.27±2.23% vs. 6.07±0.88%, N=7/each group, P<0.001) and improved functional outcome assayed by adhesive removal test (59±13.1 sec, N=7 in floxed control vs. 26.28±4.1 sec, N=12 in KO, P<0.01). Mechanistically, microglial EZH2 deletion led to a decrease in expression of M1 marker iNOS (170±14.78 vs. 76.65±11.38 cells/mm 2 , N=4/each group, P<0.05), an increase in M2 marker Arg1 (96.64±11.48 vs. 203.3±22.02 cells/mm 2 , N=4/each group, P<0.05) co-stained in microglia (Iba1). Finally, GSK343 treatment robustly reduced infarct volume (37.1±2.97% vs. 21.9±2.92%) and increased latency to fall in hang-wire test (23.5±3.5 vs. 45.9±7.1 sec) (N=8/group, p<0.05) 3 days after stroke. Conclusions: Both genetic deletion of EZH2 in microglia and pharmacological inhibition of EZH2 improved stroke outcome in aged. The effect may be due to limiting microglial M1 polarization and enhancing M2 polarization.