Journal: Bioengineering & Translational Medicine

Article Title: Neuron‐targeted 2‐deoxyglucose‐dendrimer‐rosiglitazone nanotherapy mitigates neuroinflammation and cognitive deficits in pediatric traumatic brain injury

doi: 10.1002/btm2.70053

Figure Lengend Snippet: FIGURE 8 mRNA expressions of pro- and anti-inflammatory markers and cell death markers from sham (n = 12, 6 M/6 F), traumatic brain injury (TBI) + vehicle (n = 12, 6 M/6 F), TBI + rosiglitazone (Rosi) (n = 12, 6 M/6 F), and TBI + 2-deoxyglucose (2DG-D)-Rosi (n = 12, 6 M/6 F) groups. Neurons were isolated from the injured brain regions (or the matching area from the sham mice) at 24-h post-treatment for gene expression evaluations. Data were presented as mean ± SEM. Treatment groups were presented as sham (gray circles), TBI + vehicle (blue circles), TBI + Rosi (magenta circles), and TBI + 2DG-D-Rosi (green circles). (a–d) The expression of pro-inflammatory makers, tumor necrosis factor-alpha (TNF-α) (a), interleukin-6 beta (IL-1β) (b), Toll-like receptor 4 (TLR4) (c), and NLR Family Pyrin Domain Containing 3 (NLRP3) (d). (e) The expression of anti-inflammatory maker IL-13. (f) The expression of cell death marker Fas. *p < 0.05; **p < 0.01; ***p < 0.001. (g) The co- localization of neurodegenerative markers marker Fluoro-Jade C (FJC) (green) and neuronal marker (NeuN, red) was evaluated in sham (n = 8, 4 M/4 F), TBI + vehicle (n = 8, 4 M/4 F), TBI + Rosi (n = 8, 4 M/4 F), and TBI + 2DG-D-Rosi (n = 8, 4 M/4 F) groups at 24-h post-injury. Images (40, 5 images/animal) were randomly acquired from the cortex area (mainly primary motor cortex and primary somatosensory cortex) in the injured brain regions (approximately between bregma +1 mm and bregma 0.5 mm) using the Nikon Eclipse TS2R fluorescent microscope (Nikon, NY, USA). The representative images from male treatment groups (upper panels) and female treatment groups (lower panels). 40,6-Diamidino-2-phenylindole (DAPI) (blue) was used for nucleus stains. Scale bars: 100 μm. (h) The quantification of FJC+NeuN+ cells among treatment groups. Data were presented as mean ± SEM. Treatment groups were presented as sham (gray circles), TBI + vehicle (blue circles), TBI + Rosi (magenta circles), and TBI + 2DG-D-Rosi (green circles). *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: Brain sections were washed 3 times in PBS for 5 min each, incubated in 0.001% Fluoro-Jade C (FJC) (Cat# TR-160-FJC, Biosensis, CA, USA) for 10 min, washed six times in PBS for 15 min each, and incubated with rabbit anti-NeuN (1:250; Cat# ab177487; Abcam, MA, USA).

Techniques: Isolation, Gene Expression, Expressing, Marker, Microscopy

Journal: bioRxiv

Article Title: Differential vulnerability of neuronal subpopulations of the subiculum in a mouse model for mesial temporal lobe epilepsy

doi: 10.1101/2022.06.02.494518

Figure Lengend Snippet: A-I) FluoroJade C (FJC) staining was performed to mark degenerating neurons at 2 days ( A-C ) and 4 days after KA injection ( D-I ) along the DV axis. Representative sections are shown, borders of the CA1 pyramidal cell layer are indicated. A) At 2 days after KA, FJC + cells are distributed throughout the pyramidal cell layer (superficial (s), middle (m), and deep (d) layer) of the dorsal ipsilateral subiculum. B) At the level of the injection site, FJC-stained neurons are mainly located in the superficial and middle pyramidal cell layer. Degenerating neurons are densely placed in the proximal subiculum close to CA1 and in the distal subiculum at the border to the presubiculum (arrows). C) Similar pattern in the ventral subiculum but with lower density of FJC-positive cells. D-F) At 4 days after KA the distribution of FJC + cells is similar to 2d, but less dense at all DV positions. G-I) In the contralateral subiculum, only a few neurons were marked along the DV axis. J-R) Representative horizontal sections of a fluorescent in situ hybridization for Gad67 mRNA in a control ( J-L ) and an epileptic ( M-R ) mouse 21 days after kainate (KA) injection. J) Dorsal subiculum after NaCl injection. GABAergic interneurons are equally distributed in all layers without a difference between the proximal and distal subiculum. K) At the level of the injection site, the number of Gad67 mRNA expressing cells is similar to the dorsal position. L) Ventrally the density of GABAergic interneurons is slightly higher. M-O) Ipsilateral subiculum 21 days after KA injection, dorsal position ( M ) and injection site ( N ). The density of Gad67 mRNA expressing cells is strongly decreased. O) At the ventral position more Gad67 mRNA-expressing cells were preserved than at the more dorsal sites. P-R) Contralateral subiculum 21 days after KA injection. The density of Gad67 mRNA expressing neurons is comparable to control at all DV levels but Gad67 mRNA labeling was weaker. S-U) Quantification of Gad67 mRNA expressing cells in the subiculum of NaCl-injected mice (ipsi- and contralateral side combined), ipsi- and contralateral subiculum of KA-injected mice 21 days after injection. Individual animals are color-coded, bars show means (cells/mm ) with standard error of the mean (SEM). S) In the dorsal subiculum Gad67 mRNA-expressing cells in the ipsilateral subiculum 21 days after KA injection are significantly reduced compared to controls (one-way ANOVA: p=0.003; Tukey’s multiple comparisons test: NaCl-KAi p=0.002). T) At the level of the injection site the density of Gad67 mRNA-expressing cells in the ipsilateral subiculum is significantly reduced compared to the contralateral subiculum and controls (one-way ANOVA: p=0.004; Tukey’s multiple comparisons test: NaCl-KAi p=0.004, KAc-KAi p=0.029). U) At the ventral position, density of GABAergic interneurons varies strongly across epileptic mice, means are not different (one-way ANOVA p=0.115). Scale bars: 250 μm. *p<0.05, **p<0.01. CA1 cornu ammonis 1, d deep pyramidal cell layer, m middle pyramidal cell layer, s superficial pyramidal cell layer.

Article Snippet: FluoroJade C (FJC, Merck Millipore) staining was used to monitor cell death 2 and 4 days after ihKA injection.

Techniques: Staining, Injection, In Situ Hybridization, Expressing, Labeling

Journal: Annals of Translational Medicine

Article Title: Echinocystic acid provides a neuroprotective effect via the PI3K/AKT pathway in intracerebral haemorrhage mice

doi: 10.21037/atm.2019.12.35

Figure Lengend Snippet: Echinocystic acid (EA) protected against cerebral intracerebral haemorrhage (ICH) injury by regulating the PI3K/AKT apoptosis signalling pathway. Mice were treated with EA (50 mg/kg, i.p.) or vehicle once a day for 3 consecutive days after ICH. In the ICH+EA+LY294002 group, mice were treated with the PI3K inhibitor LY294002 (i.p. 5 µL of 10 mM LY-294002 dissolved in 3% DMSO) 15 min before cerebral haemorrhage once a day for 3 consecutive days. Cerebral tissue was collected, and protein levels were determined by Western blot. (A,B) The levels of p-AKT, AKT and β-actin. (C) Fluoro-jade C (FJC) staining images in each group. There were more positive cells in the ICH+EA+LY294002 group than in the ICH+EA group. (D) This picture shows the quantitative analysis of the ratio of p-AKT to AKT. The bar graph represents the mean ± S.E.M. of 3 brains in each group, *, P<0.05 compared with the ICH group. (E) The number of damaged nerve cells in the 40× high-magnification fields is shown. Bars represent the mean ± S.E.M. of 6 brains. *, P<0.05 versus vehicle-treated ICH group.

Article Snippet: Fluoro-Jade C (FJC) was purchased from Affiliate of Merck KGaA, Darmstadt, Germany.

Techniques: Western Blot, Staining

Journal: Neural Regeneration Research

Article Title: Vav1 promotes inflammation and neuronal apoptosis in cerebral ischemia/reperfusion injury by upregulating microglial and NLRP3 inflammasome activation

doi: 10.4103/1673-5374.371368

Figure Lengend Snippet: Vav1 knockdown alleviates neuronal loss and apoptosis in the ischemic penumbra of MCAO/R rats. (A) FJC staining of the ischemic penumbra area of brain tissues in MCAO/R rats. (B) Quantification of FJC-positive cells (green). (C) Immunofluorescence staining of neurons (NeuN, FITC, green) and TUNEL (red) staining in the ischemic penumbra area in MCAO/R rats. MCAO/R rats showed greater number of TUNEL-positive cells than sham rats, but inhibition of Vav1 decreased this effect. Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (D) Quantification of NeuN and TUNEL double positive cells. (E, F) Bcl-2, Bax, and cleaved caspase-3 protein levels (normalized to the sham group) were analyzed by western blot. Data are expressed as the mean ± SD ( n = 6), and were analyzed by one-way analysis of variance followed by Tukey’s multiple comparisons test. The experiments were repeated three times. DAPI: 4′,6-Diamidino-2-phenylindole; FITC: fluorescein isothiocyanate; FJC staining: Fluoro-Jade C staining; MCAO/R: middle cerebral artery occlusion/reperfusion; NeuN: neuronal nuclear; TUNEL: terminal-deoxynucleotidyl transferase mediated nick end labeling; Vav1: Vav guanine nucleotide exchange factor 1.

Article Snippet: A Fluoro-Jade C (FJC) kit (Solarbio, G3263) was used to stain degenerating neurons according to the manufacturer’s instructions.

Techniques: Staining, Immunofluorescence, TUNEL Assay, Inhibition, Western Blot, End Labeling