p75 ntr-knockout mice  (Jackson Laboratory)

Journal: International Journal of Molecular Sciences

Article Title: Modulation of p75 NTR on Mesenchymal Stem Cells Increases Their Vascular Protection in Retinal Ischemia-Reperfusion Mouse Model

doi: 10.3390/ijms22020829

Figure Lengend Snippet: Deletion of p75 NTR reduced the development of acellular capillaries in ischemic retinas. ( A , B ) Representative trypsin-digested and PASH-stained retinas of WT and p75 NTR-/- mice subjected to I/R. Bright field imaging showed an increased number of acellular capillaries (red arrows) in WT that was attenuated in p75 NTR-/- mice (20x magnification. ( C ) Statistical analysis using two-way ANOVA showed significant impact of disease condition (ischemia) and gene deletion on the number of acellular capillaries. I/R significantly increased the number of acellular capillaries in WT retinas when compared to shams. This effect was ameliorated in retinas of p75 NTR-/- mice (*, significant using two-way ANOVA, n = 4–5).

Article Snippet: The p75 NTR , B6.129S4Ngfrtm1Jae /J (p75 NTR-/- , exon III knockout mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6-J mice (Jackson Laboratories).

Techniques: Staining, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Modulation of p75 NTR on Mesenchymal Stem Cells Increases Their Vascular Protection in Retinal Ischemia-Reperfusion Mouse Model

doi: 10.3390/ijms22020829

Figure Lengend Snippet: MSCs incorporate to ischemic retinal vasculature and decreased acellular capillaries in WT and p75 NTR-/- ischemic retinas. ( A , B ) Confocal images of retinal flat mounts stained with isolectin (red), showing incorporation of intravitreally-injected GFP-labeled MSCs (green) into WT retinas subjected to I/R (40x magnification). ( A ) Different regions of flat-mounted retina 3 days post injections showing some MSCs morphed into amoboid/satellite-like shape (green, left panel) and some MSCs picked up isolectin stain (yellow, right panel). ( B ) Different regions of flat-mounted retina 1 week post injection showing, in the left panel, that some of the MSCs appeared perivascular (green) wrapped around retinal capillaries (red) and some other MSCs (right panel) maintained satellite-like shape and co-stained with isolectin (yellow) within retinal capillaries (red), suggesting integration into retinal vasculature. ( C ) Representative trypsin-digested and PASH-stained retinas of WT and p75 NTR-/- mice subjected to I/R and receiving GFP-labeled MSCs. Bright field imaging showed decreased count for acellular capillaries (red arrows, 20x magnification). ( D ) Bar graph and statistical analysis for acellular capillaries count in WT and p75 NTR-/- retinas subjected to I/R and receiving GFP-labeled MSCs. Ischemic WT retinas still showed a significant increase in the count of acellular capillaries that was completely ameliorated in p75 NTR-/- retinas following MSCs injection (*, significant using two-way ANOVA, n = 7–11).

Article Snippet: The p75 NTR , B6.129S4Ngfrtm1Jae /J (p75 NTR-/- , exon III knockout mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6-J mice (Jackson Laboratories).

Techniques: Staining, Injection, Labeling, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Modulation of p75 NTR on Mesenchymal Stem Cells Increases Their Vascular Protection in Retinal Ischemia-Reperfusion Mouse Model

doi: 10.3390/ijms22020829

Figure Lengend Snippet: Silencing p75 NTR expression in MSCs increased vascular homing and protection in ischemic retinas. ( A ) Confocal images of ischemic WT retinal flat mounts stained with Isolectin-GS (red), showing numerous GFP-labeled MSCs (green) recruited to retinal capillaries (red) after silencing p75 NTR expression when compared to scrambled-MSCs (40x magnification). ( B ) Representative trypsin-digested and PASH-stained ischemic WT retinas showing decreased acellular capillaries (red arrows) in ischemic retinas receiving MSCs that do not express p75 NTR (20x magnification). ( C ) Bar graph and statistical analysis for acellular capillaries count in ischemic WT retinas receiving Scrambled mRNA-treated or siRNA-treated MSCs against p75 NTR . Number of acellular capillaries was significantly higher in retinas receiving p75 NTR -expressing MSCs but not p75 NTR -knocked down cells (*, significant using two-way ANOVA, n = 4).

Article Snippet: The p75 NTR , B6.129S4Ngfrtm1Jae /J (p75 NTR-/- , exon III knockout mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6-J mice (Jackson Laboratories).

Techniques: Expressing, Staining, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Modulation of p75 NTR on Mesenchymal Stem Cells Increases Their Vascular Protection in Retinal Ischemia-Reperfusion Mouse Model

doi: 10.3390/ijms22020829

Figure Lengend Snippet: Silencing p75 NTR expression improves MSCs-secretome. Representative micrograph of Western blotting ( A ) and statistical analysis ( B – D ) of GFP-MSCs CM showed 1.8-Fold increase in SDF-1-a, 6-Fold increase in VEGF-A and 5.8-Fold increase in NGF, upon silencing the expression of p75 NTR (* p < 0.05, n = 3. Significance detected by unpaired student T-test).

Article Snippet: The p75 NTR , B6.129S4Ngfrtm1Jae /J (p75 NTR-/- , exon III knockout mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6-J mice (Jackson Laboratories).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Modulation of p75 NTR on Mesenchymal Stem Cells Increases Their Vascular Protection in Retinal Ischemia-Reperfusion Mouse Model

doi: 10.3390/ijms22020829

Figure Lengend Snippet: Silencing p75 NTR expression on MSCs improves paracrine effect in HREs. RT-PCR analysis showed that treatment of HREs with CM of p75 NTR -silenced MSCs significantly increased gene expression of angiogenic and survival factor transcripts, including VEGF-A ( A ), Akt ( B ), Bcl-2 ( C ) and some of the apoptotic markers including BAX ( D ) , but no change was observed in p53 ( E ) or caspase-3 ( F ) transcripts when compared to HREs treated with CM of control cells. *, significant at p < 0.05; NS, not significant. Significance was detected by unpaired student T-test, n = 3.

Article Snippet: The p75 NTR , B6.129S4Ngfrtm1Jae /J (p75 NTR-/- , exon III knockout mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6-J mice (Jackson Laboratories).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control

Journal: International Journal of Molecular Sciences

Article Title: Modulation of p75 NTR on Mesenchymal Stem Cells Increases Their Vascular Protection in Retinal Ischemia-Reperfusion Mouse Model

doi: 10.3390/ijms22020829

Figure Lengend Snippet: Silencing p75 NTR expression on MSCs improves angiogenic response in HREs. ( A , B ) Representative images and statistical analysis of HREs migration shows significant increase in HREs migration (distance marked from original plating line marked with blue pen) after 12 h treatment with CM of p75 NTR -silenced MSCs (*, significance using one-way ANOVA test, n = 5–7. ( C – E ) Representative phase contrast images ( C ) and statistical analysis for tube length ( D ) and number of tube junction ( E ) formed in vitro by HREs after 24 h treatment with conditioned medium of p75 NTR -silenced MSCs. Results showed significant increase in tube length and number of tube junctions (*, significance using one-way ANOVA test, n = 3).

Article Snippet: The p75 NTR , B6.129S4Ngfrtm1Jae /J (p75 NTR-/- , exon III knockout mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6-J mice (Jackson Laboratories).

Techniques: Expressing, Migration, In Vitro

Journal: International Journal of Molecular Sciences

Article Title: Modulation of p75 NTR on Mesenchymal Stem Cells Increases Their Vascular Protection in Retinal Ischemia-Reperfusion Mouse Model

doi: 10.3390/ijms22020829

Figure Lengend Snippet: Modulating p75 NTR on MSCs using LM11A-31 improved their secretome and improved paracrine effect in HREs. Representative Western blotting ( A ) and statistical analysis of MSCs secretome treated with the pharmacologic modulator of p75 NTR ; LM11A-31. ( B – D ) showing ~14.9-Fold increase in SDF-1a ( B ), 1.6-Fold increase in VEGF-A ( C ) and ~4-Fold increase in NGF ( D ). * p < 0.05, Significance was detected by unpaired Student t-test, n = 3. RT-PCR analysis showed that treatment of HREs with CM of LM11A-31-treated MSCs significantly increased gene expression of the survival factor transcripts VEGF-A ( E ), Akt ( F ), Bcl-2 ( G ) and some of the apoptotic markers, such as Bax ( H ), but not p53 ( I ). *, Significant at p < 0.05; NS, not significant. Significance was detected by unpaired Student t-test, n = 3.

Article Snippet: The p75 NTR , B6.129S4Ngfrtm1Jae /J (p75 NTR-/- , exon III knockout mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6-J mice (Jackson Laboratories).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Modulation of p75 NTR on Mesenchymal Stem Cells Increases Their Vascular Protection in Retinal Ischemia-Reperfusion Mouse Model

doi: 10.3390/ijms22020829

Figure Lengend Snippet: The sequence of the PCR primers used in the mRNA quantification experiments.

Article Snippet: The p75 NTR , B6.129S4Ngfrtm1Jae /J (p75 NTR-/- , exon III knockout mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6-J mice (Jackson Laboratories).

Techniques: Sequencing

Journal: Scientific Reports

Article Title: Deletion of p75 NTR prevents vaso-obliteration and retinal neovascularization via activation of Trk- A receptor in ischemic retinopathy model

doi: 10.1038/s41598-018-30029-0

Figure Lengend Snippet: Hyperoxia, but not hypoxia triggered p75 NTR expression in WT-pups in OIR model. ( A ) Exposing WT pups to 70% oxygen from p7-p12 significantly increased p75 NTR expression in WT-pups (*significant compared to WT-controls using two-way ANOVA, p < 0.05, n = 3–4). ( B ) Western blotting representative and bar graph of p75 NTR receptor expression in WT pups by p14 showing no alteration during hypoxic stage of OIR (n = 4–6). ( C ) Western blotting representative and bar graph of p75 NTR receptor expression in WT pups by p17 showing a trend for decreased p75 NTR expression that was not statistically significant (n = 4).

Article Snippet: The p75 NTR , B6.129S4Ngfr tm1Jae /J (p75 NTR+/− , exon III knockout mice ) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6/J mice (Jackson Laboratories).

Techniques: Expressing, Western Blot

Journal: Scientific Reports

Article Title: Deletion of p75 NTR prevents vaso-obliteration and retinal neovascularization via activation of Trk- A receptor in ischemic retinopathy model

doi: 10.1038/s41598-018-30029-0

Figure Lengend Snippet: Deletion of p75 NTR attenuated hyperoxia-induced increase in proNGF and decrease in NGF. ( A ) Representative Western blotting and scatter graph showing significant increase in proNGF protein levels in retinas from WT, but not in p75 NTR−/− in response to hyperoxia. (*significant compared to the rest of the groups using two-way ANOVA, p < 0.05, n = 6–8). ( B ) Representative Western blotting and scatter graph showing a tendency of NGF protein level to be decreased in WT (P = 0.07), but preserved-level in p75 NTR−/− during hyperoxia. ( C ) Bar graph showing significant decrease in of NGF/proNGF ratio in retinas from WT, but not p75 NTR−/− pups in response to hyperoxia (*significant compared to WT normoxic controls, using two-way ANOVA, p < 0.05, n = 6–8).

Article Snippet: The p75 NTR , B6.129S4Ngfr tm1Jae /J (p75 NTR+/− , exon III knockout mice ) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6/J mice (Jackson Laboratories).

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Deletion of p75 NTR prevents vaso-obliteration and retinal neovascularization via activation of Trk- A receptor in ischemic retinopathy model

doi: 10.1038/s41598-018-30029-0

Figure Lengend Snippet: Deletion of p75 NTR preserved survival signal and attenuated hyperoxia-mediated apoptosis. ( A ) Representative Western blotting and bar graph showing modest decrease in Akt activation (Y308) in response to hyperoxia at p12 retinal lysates of WT and p75 NTR−/− mice (n = 5–7). ( B ) Representative Western blotting and bar graph of total PARP expression in p12 retinal lysates of WT and p75 NTR−/− mice (*significant using two-way ANOVA, p < 0.05, n = 7). ( C ) Representative Western blotting and bar graph of cleaved PARP expression in p12 retinal lysates of WT and p75 NTR−/− mice (*significant using two-way ANOVA, p < 0.05, n = 4–6).

Article Snippet: The p75 NTR , B6.129S4Ngfr tm1Jae /J (p75 NTR+/− , exon III knockout mice ) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6/J mice (Jackson Laboratories).

Techniques: Western Blot, Activation Assay, Expressing

Journal: Scientific Reports

Article Title: Deletion of p75 NTR prevents vaso-obliteration and retinal neovascularization via activation of Trk- A receptor in ischemic retinopathy model

doi: 10.1038/s41598-018-30029-0

Figure Lengend Snippet: Deletion of p75 NTR attenuated hyperoxia-induced retinal central vaso-obliteration. ( A , B ) Representative images of Isolectin GS-stained retinal flat mounts of WT and p75 NTR−/− pups showing capillary drop-out (CDO) illustrated by encircled area at the center at p12 (magnification is 5X). ( C ) Bar graph and statistical analysis using unpaired student T-test for CDO showing significant reduction of CDO in p75 NTR−/− compared to WT pups in response to hyperoxia (*significant compared to WT-hyperoxic group, p < 0.05, n = 8–12). (Hyper) hyperoxia-exposed group, (Norm) normoxic controls, (CDO): central capillary dropout.

Article Snippet: The p75 NTR , B6.129S4Ngfr tm1Jae /J (p75 NTR+/− , exon III knockout mice ) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6/J mice (Jackson Laboratories).

Techniques: Staining

Journal: Scientific Reports

Article Title: Deletion of p75 NTR prevents vaso-obliteration and retinal neovascularization via activation of Trk- A receptor in ischemic retinopathy model

doi: 10.1038/s41598-018-30029-0

Figure Lengend Snippet: Deletion of p75 NTR attenuated hypoxia-induced neovascularization and enhances reparative angiogenesis. ( A , B ) Representative images of GS-isolectin stained retinal flat mounts of WT and p75 NTR−/− pups at p17, showing pathological retinal neovascularization (RNV, encircled by dotted white line) and central avascular area (encircled area at the center). Images were taken on 5X magnification. Bar graph of RNV ( C ) and central avascular area ( D ) showing significant decrease in both manifestations in p75 NTR−/− pups by p14 during hypoxic stage of OIR (*significant using unpaired student T-test, p < 0.05, n = 7–14). Hypo: relative hypoxia-exposed groups, RNV: retinal neovascularization.

Article Snippet: The p75 NTR , B6.129S4Ngfr tm1Jae /J (p75 NTR+/− , exon III knockout mice ) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6/J mice (Jackson Laboratories).

Techniques: Staining

Journal: Scientific Reports

Article Title: Deletion of p75 NTR prevents vaso-obliteration and retinal neovascularization via activation of Trk- A receptor in ischemic retinopathy model

doi: 10.1038/s41598-018-30029-0

Figure Lengend Snippet: Deletion of p75 NTR preserved hypoxia-induced VEGF expression and VEGFR2 activation. ( A ) Representative Western blotting and bar graph of retinal VEGF protein expression from WT and p75 NTR−/− pups exposed to OIR at p14. Two-way ANOVA showed significant effect of hypoxia increasing VEGF both in WT and p75 NTR−/− pups (*significant compared to WT normoxic group, p < 0.05, n = 6–12). ( B ) Representative Western blotting and bar graph of pVEGFR2 compared to its total protein VEGFR2 level. Two-way ANOVA showed marked gene deletion effect where p75 NTR−/− showed significant increase in pVEGFR2 under normoxia or hypoxia compared to WT-normoxic controls (*significant compared to WT normoxic group using, p < 0.05, n = 4–5).

Article Snippet: The p75 NTR , B6.129S4Ngfr tm1Jae /J (p75 NTR+/− , exon III knockout mice ) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6/J mice (Jackson Laboratories).

Techniques: Expressing, Activation Assay, Western Blot

Journal: Scientific Reports

Article Title: Deletion of p75 NTR prevents vaso-obliteration and retinal neovascularization via activation of Trk- A receptor in ischemic retinopathy model

doi: 10.1038/s41598-018-30029-0

Figure Lengend Snippet: Deletion of p75 NTR attenuated hypoxia-induced increase in proNGF and restored ratio of NGF/proNGF. ( A ) Representative Western blotting and scatter graph for proNGF showing that hypoxia caused significant increase in proNGF levels in retinas from WT-pups but not p75 NTR−/− compared to normoxic control at p14. ( B ) Representative Western blotting and scatter graph for NGF showing that hypoxia caused significant increase in NGF level in retinas from p75 NTR−/− , but not WT compared to their normoxic controls. ( C ) Bar graph of NGF/proNGF ratio and Two-way ANOVA statistical analysis showing significant decrease in retinal NGF/proNGF ratio in WT-pups during hypoxia while p75 NTR−/− pups maintained balanced ratio of NGF/proNGF (*significant using Two-way ANOVA, p < 0.05, n = 5–7).

Article Snippet: The p75 NTR , B6.129S4Ngfr tm1Jae /J (p75 NTR+/− , exon III knockout mice ) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6/J mice (Jackson Laboratories).

Techniques: Western Blot, Control

Journal: Scientific Reports

Article Title: Deletion of p75 NTR prevents vaso-obliteration and retinal neovascularization via activation of Trk- A receptor in ischemic retinopathy model

doi: 10.1038/s41598-018-30029-0

Figure Lengend Snippet: Deletion of p75 NTR enhanced expression and activation of NGF survival receptor; TrkA. ( A ) Quantitative real-time PCR of TrkA gene expression in p12 WT and p75 NTR−/− exposed to OIR showing significant increase in basal mRNA expression in p75 NTR−/− retinas compared to the other groups (*significant using Two-Way ANOVA, p < 0.05, n = 6). ( B , C ) Representative Western blotting and bar graph statistical analysis of TrkA expression by p12 showing significant gene effect and significant increase in TrkA expression in p75 NTR−/− pups compared to their corresponding WT-controls under normoxia and hyperoxia (*significant using Two-way ANOVA, p < 0.05, n = 6–9). ( B , D ) Representative Western blotting and bar graph statistical analysis of pTrkA activation by p12 showing an overall significant gene effect and a significant increase in pTrkA activation (Y490) in p75 NTR−/− pups exposed to hyperoxia compared to the rest of the groups (*significant using Two-way ANOVA, p < 0.05, n = 6–9). ( E , F ) Representative Western blotting and bar graph statistical analysis of pTrkA activation by p14 showing an overall significant effect of hypoxia ( # ) to increase activation of pTrkA (Y490) in p75NTR −/− pups, however there was no significant difference between individual groups (n = 6–9).

Article Snippet: The p75 NTR , B6.129S4Ngfr tm1Jae /J (p75 NTR+/− , exon III knockout mice ) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6/J mice (Jackson Laboratories).

Techniques: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot

Journal: Scientific Reports

Article Title: Deletion of p75 NTR prevents vaso-obliteration and retinal neovascularization via activation of Trk- A receptor in ischemic retinopathy model

doi: 10.1038/s41598-018-30029-0

Figure Lengend Snippet: TrkA activity is required for the vaso-protective effects observed in p75 NTR−/− pups. (A , B) Representatives of p17 retinal flat mounts of FITC-perfused p75 NTR−/− pups exposed to OIR and receiving DMSO or K252a (0.5 μg μL −1 /eye) showing central avascular area encircled in the center by white line, 5X magnification. (C) Statistical analysis of central capillary dropout (CDO) area showing significant increase of CDO in p75 NTR−/− pups that received intravitreal injection of K252a compared to DMSO-receiving p75 NTR−/− group or WT-pups that received K252a. There was no significant difference between WT-pups that received K252a compared to DMSO injection. (*Significant using Two-Way ANOVA, p < 0.05, n = 5–7). ( D , E ) Representatives of the same p75 NTR−/− retinal flat mounts stained with GS-isolectin to illustrate retinal neovascularization (RNV) at retinal mid-periphery (encircled by dotted white line, 5X magnification). ( F ) Statistical analysis using two-way ANOVA showing significant increase of RNV in p75 NTR−/− pups that received intravitreal injection of K252a compared to DMSO-injected WT or p75 NTR−/− groups. There was no significant difference between WT-pups that received K252a compared to DMSO injection. (*Significant using Two-Way ANOVA, p < 0.05, n = 4–7).

Article Snippet: The p75 NTR , B6.129S4Ngfr tm1Jae /J (p75 NTR+/− , exon III knockout mice ) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6/J mice (Jackson Laboratories).

Techniques: Activity Assay, Injection, Staining

Journal: Journal of diabetes, metabolic disorders & control

Article Title: Deletion of the Neurotrophin Receptor p75 NTR Prevents Diabetes-Induced Retinal Acellular Capillaries in Streptozotocin-Induced Mouse Diabetic Model

doi: 10.15406/jdmdc.2017.04.00129

Figure Lengend Snippet: A summary of sources of antibodies used to detect protein expression using Western Blot.

Article Snippet: Induction of diabetes Male 8-week old littermates of wild-type and p75 NTR knockout mice (~25g) from our colony with founders originally purchased from Jackson laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, Western Blot

Journal: Journal of diabetes, metabolic disorders & control

Article Title: Deletion of the Neurotrophin Receptor p75 NTR Prevents Diabetes-Induced Retinal Acellular Capillaries in Streptozotocin-Induced Mouse Diabetic Model

doi: 10.15406/jdmdc.2017.04.00129

Figure Lengend Snippet: Genetic deletion of p75NTR mitigated diabetes-induced acellular capillaries. (C–F): Representative images of trypsinized-retina from WT or p75NTR KO controls or diabetic mice. (G): Quantitative data showing significant increase in retinal acellular capillaries after 6-month of diabetes in WT-diabetic but not in p75 KO-diabetic animals compared to respective controls. (A–B): Representative images of a typical occluded (acellular) capillary in a trypsinized retina of diabetic animals double-labelled with anti-Collagen IV (Green) and CD31 (Red).

Article Snippet: Induction of diabetes Male 8-week old littermates of wild-type and p75 NTR knockout mice (~25g) from our colony with founders originally purchased from Jackson laboratory (Bar Harbor, ME, USA).

Techniques:

Journal: Molecular Brain

Article Title: Multiple functions of precursor BDNF to CNS neurons: negative regulation of neurite growth, spine formation and cell survival

doi: 10.1186/1756-6606-2-27

Figure Lengend Snippet: proBDNF reduces dendritic spine density in hippocampal neurons . Cultured neurons were maintained for 3–4 weeks and treated with the indicated reagents (50 ng/ml) for 2 (A, B, and D) or 3 days (C). In A and B, neurons were cultured in serum-containing medium. (A) CR-proBDNF reduced the density of DiI-labeled spines. Representative images of DiI-labeled neurons treated with the indicated reagents for 2 days (left). Summary of spine density (middle) and length (right). Data were collected from 52 (Mock), 59 (matBDNF), and 38 (CR-proBDNF) independent cells (non-parametric test, * P < 0.05, compared to Mock). Note that matBDNF increased spine density, while proBDNF reduced spine density (arrows and arrowheads). (B) proBDNF promoted the shrinkage of phalloidin-labeled spines and p75 NTR was involved in the proBDNF action. Application of a functionally blocking antibody against p75 NTR , AB1554 was performed and fixed neurons were stained with an anti-MAP2 antibody and FITC-labeled phalloidin. Representative images of the double-stained neurons (left) and a quantitative analysis of phalloidin-labeled spiny structures (right) are shown, n = 30–40 independent cells from three independent coverslips. t -test, * P < 0.05, ** P < 0.01, compared to Mock (100% as control). (C) CR-proBDNF decreases spine density in low-density hippocampal neurons cultured in serum-free medium. The cultures were incubated with CR-proBDNF in serum-free medium for 2 days and double-stained with an anti-MAP2 antibody and FITC-labeled phalloidin. n = 24–32 cells from three independent coverslips. t -test, * P < 0.05, ** P < 0.01, compared to Mock. (D) CR-proBDNF decreased spine density in hippocampal slices. Representative images of DiI-labeled neurons at low and high magnification are shown (left). A decrease in spine density was observed in CR-proBDNF-treated slices (arrow heads). Quantification of spine density (right). Data were collected from 7 (Mock) and 6 (CR-proBDNF) independent slices. Note that CR-proBDNF-treated slices showed a decrease in spine density when compared with matBDNF. t -test, * P < 0.05. Scale bar, 10 μm (A, B, and D).

Article Snippet: The p75 NTR knockout mice (ngfr tm1Jae ) were from The Jackson Laboratory (Bar Harbor, ME).

Techniques: Cell Culture, Labeling, Blocking Assay, Staining, Control, Incubation