Control of Axonal Growth and Regeneration of Sensory Neurons by the p110δ PI 3-Kinase methods
Aim. Evidence-backed execution summary for Control of Axonal Growth and Regeneration of Sensory Neurons by the p110δ PI 3-Kinase methods from Control of Axonal Growth and Regeneration of Sensory Neurons by the p110δ PI 3-Kinase.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Inactivation of p110δ increases the vulnerability of sensory neurons to growth cone collapse and decreases axona...
reagent used in the protocol.
- Use
- We next assessed the responsiveness of neurons to PI3K inhibition using the pan-PI3K inhibitor LY294002, which induces growth cone collapse in sensory neurons,. Our analyses showed that this response was significantly greater in p110δ KI than in WT DRG neurons ( ). This indicates that DRG neurons with inacti...
Inactivation of p110δ increases the vulnerability of sensory neurons to growth cone collapse and decreases axona...
reagent used in the protocol.
- Use
- (A) Growth cone collapse in DRG explants induced by LY294002 (a pan-PI3K inhibitor) or IC87114 (a p110δ-selective inhibitor). Both drugs were used at 10 µM for 10 min ( n ≥6 independent experiments). *p<0.01. (B) E13.5 DRG explants were cultured for 24 h on 20 µg/ml laminin and treated with Sema...
Reduced Akt and increased RhoA/PTEN signaling in neurons with inactive p110δ
reagent used in the protocol.
- Use
- p110δ has recently been demonstrated to inhibit the activity of the tumor suppressor PTEN through a pathway involving RhoA. Similary, in p110δ KI DRGs and brain homogenates, PTEN lipid phosphatase activity was constitutively elevated ( ). In addition, GTP-loading of RhoA, but not Rac, was significantly in...
Embryo and tissue preparation
reagent used in the protocol.
- Use
- p110α lz and p110δ lz mice were transferred into ice-cold PBS, and fixed for 1 h on ice in fixing buffer (4% paraformaldehyde/0.2% glutaraldehyde/2 mM MgCl 2 /5 mM EGTA/0.02% NP40). Embryos were then washed 3 times in washing buffer (PBS/2 mM MgCl 2 /0.01% sodium deoxycholate/0.02% NP40/5 mM EGTA) and post...
Immunohistochemistry
reagent used in the protocol.
- Use
- For stainings of DRG neurons in situ, E13.5 WT and p110δ KI mice were paraformaldehyde fixed, embedded in 20% gelatin and vibratome-sectioned at 80 µm. Anti-P-p70S6K (Cell Signaling Technology) was applied at 1∶300 overnight at 4°C. Following extensive washes, bound antibodies were detected usi...
Neuronal cultures
reagent used in the protocol.
- Use
- Age-matched WT and p110δ KI embryos were isolated at the appropriate age, and transferred into ice-cold DMEM. DRGs were dissected from E13.5 mouse embryos and extramesenchymal tissue was removed using a sharpened 0.2 mm tungsten wire. DRGs were then plated on glass coverslips previously coated with poly-L-lysin...
Immunocytochemistry
reagent used in the protocol.
- Use
- Neuronal cultures were treated as indicated and paraformaldehyde-fixed (4% paraformaldehyde/PBS/10% sucrose) for 30 min before permeabilization for 5 min with PBS/1% Triton × 100. Neurons were then labeled with Phalloidin-Alexa488 (1∶50 in PBST) and anti-βIII-tubulin antibody (1∶800; Covance). F...
SDS-PAGE and Western blotting
reagent used in the protocol.
- Use
- Dissociated neurons were washed twice with ice-cold PBS lysed for 30 min in ice-cold lysis buffer (10 mM Tris.HCl pH 7.4, 250 mM sucrose, 10 mM MgCl 2, 0.5% NP40, complete protease inhibitor cocktail (Roche), 2 mM sodium orthovanadate, 0.1 mM DTT, 25 mM NaF). All cell lysates was adjusted to equal concentrations, a...
Inactivation of p110δ increases the vulnerability of sensory neurons to growth cone collapse and decreases axona...
To determine the contribution of p110δ to neuronal development and function, we analyzed mice in which p110δ was inactivated as a result of the introduction of a germline point mutation which renders the kinase inactive (p110δ D910A;. Homozygous p110δ D910A/D910A mice, hereafter referred to as...
- Use
- To determine the contribution of p110δ to neuronal development and function, we analyzed mice in which p110δ was inactivated as a result of the introduction of a germline point mutation which renders the kinase inactive (p110δ D910A;. Homozygous p110δ D910A/D910A mice, hereafter referred to as...
Inhibition of axonal regeneration correlates with impaired functional recovery
We next assessed whether the anatomical evidence for p110δ-dependency for optimal axonal regeneration correlated with functional recovery using an automated quantitative gait analysis system, the CatWalk, to assess recovery of locomotion following nerve injury on days 1, 3, 7, 10, 14 and 21. Prior to injury, g...
- Use
- We next assessed whether the anatomical evidence for p110δ-dependency for optimal axonal regeneration correlated with functional recovery using an automated quantitative gait analysis system, the CatWalk, to assess recovery of locomotion following nerve injury on days 1, 3, 7, 10, 14 and 21. Prior to injury, g...
Inhibition of axonal regeneration correlates with impaired functional recovery
Paw pressure intensity during continuous locomotion was assessed using the CatWalk quantitative gait analysis system. Recovery in locomotion was analyzed on days 1, 3, 7, 10, 14 and 21 post-injury. (A) Intensity profiles of 5 steps during a single run from each paw, 7 days post-injury. Each intensity profile shows t...
- Use
- Paw pressure intensity during continuous locomotion was assessed using the CatWalk quantitative gait analysis system. Recovery in locomotion was analyzed on days 1, 3, 7, 10, 14 and 21 post-injury. (A) Intensity profiles of 5 steps during a single run from each paw, 7 days post-injury. Each intensity profile shows t...
The CatWalk
The CatWalk gait analysis system was used to assess functional recovery of locomotion following sciatic nerve crush injury. The animal traversed a meter long walkway with a glass floor and 2 perspex walls spaced 8 cm apart, housed in a darkened room. Light from 2 encased white fluorescent tubes entered the glass fl...
- Use
- The CatWalk gait analysis system was used to assess functional recovery of locomotion following sciatic nerve crush injury. The animal traversed a meter long walkway with a glass floor and 2 perspex walls spaced 8 cm apart, housed in a darkened room. Light from 2 encased white fluorescent tubes entered the glass fl...
Morris water maze
Mice for behavioral testing were housed in groups of 2 to 4 in individually-ventilated cages, with food and water ad libitum and maintained on a 12 h light-dark cycle. Mice tested were males between the ages of 2 and 6 months. The Morris water maze protocol has been described previously. Briefly, the mice were trai...
- Use
- Mice for behavioral testing were housed in groups of 2 to 4 in individually-ventilated cages, with food and water ad libitum and maintained on a 12 h light-dark cycle. Mice tested were males between the ages of 2 and 6 months. The Morris water maze protocol has been described previously. Briefly, the mice were trai...
Immunohistochemistry
For stainings of DRG neurons in situ, E13.5 WT and p110δ KI mice were paraformaldehyde fixed, embedded in 20% gelatin and vibratome-sectioned at 80 µm. Anti-P-p70S6K (Cell Signaling Technology) was applied at 1∶300 overnight at 4°C. Following extensive washes, bound antibodies were detected usi...
- Use
- For stainings of DRG neurons in situ, E13.5 WT and p110δ KI mice were paraformaldehyde fixed, embedded in 20% gelatin and vibratome-sectioned at 80 µm. Anti-P-p70S6K (Cell Signaling Technology) was applied at 1∶300 overnight at 4°C. Following extensive washes, bound antibodies were detected usi...
SDS-PAGE and Western blotting
Dissociated neurons were washed twice with ice-cold PBS lysed for 30 min in ice-cold lysis buffer (10 mM Tris.HCl pH 7.4, 250 mM sucrose, 10 mM MgCl 2, 0.5% NP40, complete protease inhibitor cocktail (Roche), 2 mM sodium orthovanadate, 0.1 mM DTT, 25 mM NaF). All cell lysates was adjusted to equal concentrations, a...
- Use
- Dissociated neurons were washed twice with ice-cold PBS lysed for 30 min in ice-cold lysis buffer (10 mM Tris.HCl pH 7.4, 250 mM sucrose, 10 mM MgCl 2, 0.5% NP40, complete protease inhibitor cocktail (Roche), 2 mM sodium orthovanadate, 0.1 mM DTT, 25 mM NaF). All cell lysates was adjusted to equal concentrations, a...
Supporting Information
Normal spatial memory development of p110δ KI mice in the Morris water maze. (A) WT and p110δ KI mice were trained with 12 trials per day in blocks of 4 trials. The time to reach the hidden platform is shown; there was no difference between the genotypes. (B) After training, day 3 and 5 probe trials were p...
- Use
- Normal spatial memory development of p110δ KI mice in the Morris water maze. (A) WT and p110δ KI mice were trained with 12 trials per day in blocks of 4 trials. The time to reach the hidden platform is shown; there was no difference between the genotypes. (B) After training, day 3 and 5 probe trials were p...
The CatWalk
Software used for acquisition, scoring, statistics, or reporting.
- Use
- The CatWalk gait analysis system was used to assess functional recovery of locomotion following sciatic nerve crush injury. The animal traversed a meter long walkway with a glass floor and 2 perspex walls spaced 8 cm apart, housed in a darkened room. Light from 2 encased white fluorescent tubes entered the glass fl...
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Inactivation of p110δ increases the vulnerability of sensory neurons to growth cone collapse and decreases axona...
(A) Growth cone collapse in DRG explants induced by LY294002 (a pan-PI3K inhibitor) or IC87114 (a p110δ-selective inhibitor). Both drugs were used at 10 µM for 10 min ( n ≥6 independent experiments). *p<0.01. (B) E13.5 DRG explants were cultured for 24 h on 20 µg/ml laminin and treated with Sema3A (0.3 µg/ml) for 30 min. Increased growth cone collapse in response to Sema3A (0.3 µg/ml) in p110δ KI DRGs (right). Each data point represents the %±SEM ( n ≥3 independent experiments). In each experiment at least 80 growth cones were counted. *p<0.02. (C) Example of DRG explants in each group, which have been stained for βIII-tubulin ( red ) and phalloidin ( green ). Scale bar, 20 µm.
Reduced axonal regeneration in the injured sciatic nerve of mice with inactive p110δ
Given that the absence of p110δ activity limits axonal outgrowth in embryonic neurons, we assessed the axonal growth potential of adult p110δ KI neurons. Adult peripheral nerve axons are capable of functional regeneration in mammals, with a crush injury of the sciatic nerve being a well-established injury model to study axonal growth. On day 3, extension of regenerating neuronal fibers 2 mm distal from the injury site was reduced in p110δ KI mice ( ), without any differences in the presence of cytoskeletal breakdown products 4 mm distally from the site of injury (data not shown), indicating that loss of p110δ activity does not alter normal axonal degeneration. p110δ activity has been shown to be essential for CSF-1-driven in vitro chemotaxis of macrophages,, which might affect axonal regeneration due to impaired inflammatory response at the injury site in p...
Reduced axonal regeneration in the injured sciatic nerve of mice with inactive p110δ
3 days post-injury, the sciatic nerves of WT and p110δ KI mice were fixed and cryo-sectioned. (A) Average relative fluorescence intensity profile of anti-βIII-tubulin labeling across a one-pixel line along the entire nerve segment, following cropping of the micrographs to a fixed pixel segment. (B) High-power micrographs of the sciatic nerve segments 2 mm distal to the injury, labeled with anti-βIII-tubulin (left panels and green in right panels), anti-F4/80 ( red ) and Hoechst ( blue ). Scale bar, 50 µm. (C) At 7 days post injury, L4 DRGs of WT and p110δ KI mice were fixed, vibratome-sectioned, and co-labeled with ATF3 (red) and SPRR1A (green). Percentage of SPRR1A and ATF3 co-labeling over ATF3-only positive DRG neurons in WT and p110δ KI mice. Data are from 3 WT and 4 p110δ KI mice, and are presented as mean±SEM. *p<0.05. Scale bar, 50 µm.
Inhibition of axonal regeneration correlates with impaired functional recovery
We next assessed whether the anatomical evidence for p110δ-dependency for optimal axonal regeneration correlated with functional recovery using an automated quantitative gait analysis system, the CatWalk, to assess recovery of locomotion following nerve injury on days 1, 3, 7, 10, 14 and 21. Prior to injury, gait analysis assessed by the CatWalk on a number of locomotor parameters revealed no differences between WT and p110δ KI mice (, ). In contrast, following unilateral injury to the sciatic nerve, p110δ KI mice showed a significant decrease in the recovery of the ability to bear weight on the injured paw ( ). Whilst both WT and p110δ KI mice display functional recovery during the first 10 days post-lesion, the relative paw pressure intensity in p110δ KI mice was significantly lower in comparison to WT mice (; p<0,001, 2-way repeated measured ANOVA). Thes...
Inhibition of axonal regeneration correlates with impaired functional recovery
Paw pressure intensity during continuous locomotion was assessed using the CatWalk quantitative gait analysis system. Recovery in locomotion was analyzed on days 1, 3, 7, 10, 14 and 21 post-injury. (A) Intensity profiles of 5 steps during a single run from each paw, 7 days post-injury. Each intensity profile shows two traces, a higher trace of the maximal relative intensity and a lower trace of the average relative intensity. (B) Reduced relative paw pressure intensity for each paw during recovery in p110δ KI mice compared to WT mice. Data presented is the mean±SEM of ≥6 animals. p<0.001, 2-way repeated measures ANOVA.
Embryo and tissue preparation
p110α lz and p110δ lz mice were transferred into ice-cold PBS, and fixed for 1 h on ice in fixing buffer (4% paraformaldehyde/0.2% glutaraldehyde/2 mM MgCl 2 /5 mM EGTA/0.02% NP40). Embryos were then washed 3 times in washing buffer (PBS/2 mM MgCl 2 /0.01% sodium deoxycholate/0.02% NP40/5 mM EGTA) and post-fixed in 4% paraformaldehyde for 1 h. β-galactosidase expression was visualized by incubation in X-gal developing buffer (PBS/5 mM K 3 Fe(CN) 6 /5 mM K 4 (CN) 6 /2 mM MgCl 2 /0.01% sodium deoxycholate/0.02% NP40, 1 mg/ml X-gal) overnight at room temperature. Embryos were then washed in PBS, post-fixed in 4% paraformaldehyde for 1 h, and washed twice in distilled water before dehydration though a series of 70% ethanol, 95% ethanol, and 100% ethanol. Following incubation in methylsalicylate for 1 h, embryos were rehydrated in 100% ethanol, 95% ethanol, 70% ethanol, and...
Sciatic nerve crush
For in vivo regeneration studies, 6 to 8 week-old male mice were used. The mice were assessed before surgery to establish baseline-walking patterns. Animals were anesthetized with a mixture of medetomidine (0.5 mg/kg) and ketamine (75 mg/kg), and the left sciatic nerve was exposed. A crush injury was performed 10 mm distal to the obturator tendon using forceps compression (10 sec) and the crush site labeled with lamp black. The muscle and skin layers were sutured and animals were allowed to recover in the cage post-operatively.
The CatWalk
The CatWalk gait analysis system was used to assess functional recovery of locomotion following sciatic nerve crush injury. The animal traversed a meter long walkway with a glass floor and 2 perspex walls spaced 8 cm apart, housed in a darkened room. Light from 2 encased white fluorescent tubes entered the glass floor through the distal edge of the glass, and was totally internally reflected. Light scatters only where a paw contacts the glass, illuminating the area of paw contact. This reflected light was captured using videocamera (Sentech 705, 8.5 mm, f = 1.4, variable focus and variable iris) equipped with a wide-angle objective and a frame grabber (Matrix Vision SG-board) connected to a PC running the CatWalk 500 software for capture and analysis. Each mouse ran across the CatWalk one day before surgery to establish baseline locomotor parameters. Following surgery, a...
Measurement outputs
What raw and processed outputs should exist?
Expression of class IA PI3K proteins in the hippocampus of p110δ KI mice. Tissue extracts from the hippocampus from adult WT and p110δ KI mice were immunoblotted with...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
To determine the contribution of p110δ to neuronal development and function, we analyzed mice in which p110δ was inactivated as a result of the introduction of a germl...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
We next assessed whether the anatomical evidence for p110δ-dependency for optimal axonal regeneration correlated with functional recovery using an automated quantitative ga...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Paw pressure intensity during continuous locomotion was assessed using the CatWalk quantitative gait analysis system. Recovery in locomotion was analyzed on days 1, 3, 7, 10, 14...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture run-level gait data for each animal and preserve the timepoint or treatment labeling.
inferred from protocolPreprocessing / cleaning
(A) Growth cone collapse in DRG explants induced by LY294002 (a pan-PI3K inhibitor) or IC87114 (a p110δ-selective inhibitor).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Expression of class IA PI3K proteins in the hippocampus of p110δ KI mice. Tissue extracts from the hippocampus from adult WT and p110δ KI mice were immunoblotted with...; To determine the contribution of p110δ to neuronal development and function, we analyzed mice in which p110δ was inactivated as a result of the introduction of a germl...; We next assessed whether the anatomical evidence for p110δ-dependency for optimal axonal regeneration correlated with functional recovery using an automated quantitative ga...; Paw pressure intensity during continuous locomotion was assessed using the CatWalk quantitative gait analysis system. Recovery in locomotion was analyzed on days 1, 3, 7, 10, 14....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
(A) Growth cone collapse in DRG explants induced by LY294002 (a pan-PI3K inhibitor) or IC87114 (a p110δ-selective inhibitor). Both drugs were used at 10 µM for 10 min...; Left panel, example of E13.5 DRG neurons derived from WT and p110δ KI mice, cultured on 10 µg/ml laminin for 24 h. Right panel, relative axonal length in p110δ K...; Given that the absence of p110δ activity limits axonal outgrowth in embryonic neurons, we assessed the axonal growth potential of adult p110δ KI neurons. Adult periphe...; 3 days post-injury, the sciatic nerves of WT and p110δ KI mice were fixed and cryo-sectioned. (A) Average relative fluorescence intensity profile of anti-βIII-tubulin...
from paperReporting output
Report representative outputs alongside summary comparisons for Expression of class IA PI3K proteins in the hippocampus of p110δ KI mice. Tissue extracts from the hippocampus from adult WT and p110δ KI mice were immunoblotted with..., To determine the contribution of p110δ to neuronal development and function, we analyzed mice in which p110δ was inactivated as a result of the introduction of a germl..., We next assessed whether the anatomical evidence for p110δ-dependency for optimal axonal regeneration correlated with functional recovery using an automated quantitative ga..., Paw pressure intensity during continuous locomotion was assessed using the CatWalk quantitative gait analysis system. Recovery in locomotion was analyzed on days 1, 3, 7, 10, 14....
inferred from protocolStructured statistical methods
(A) Growth cone collapse in DRG explants induced by LY294002 (a pan-PI3K inhibitor) or IC87114 (a p110δ-selective inhibitor). Both drugs were used at 10 µM for 10 min...; Left panel, example of E13.5 DRG neurons derived from WT and p110δ KI mice, cultured on 10 µg/ml laminin for 24 h. Right panel, relative axonal length in p110δ K...; Given that the absence of p110δ activity limits axonal outgrowth in embryonic neurons, we assessed the axonal growth potential of adult p110δ KI neurons. Adult periphe...; 3 days post-injury, the sciatic nerves of WT and p110δ KI mice were fixed and cryo-sectioned. (A) Average relative fluorescence intensity profile of anti-βIII-tubulin...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
(A) Growth cone collapse in DRG explants induced by LY294002 (a pan-PI3K inhibitor) or IC87114 (a p110δ-selective inhibitor). Both drugs were used at 10 µM for 10 min ( n ≥6 independent experiments). *p<0.01. (B) E13.5 DRG explants were cultured for 24 h on 20 µg/ml laminin and treated with Sema3A (0.3 µg/ml) for 30 min. Increased growth cone collapse in response to Sema3A (0.3 µg/ml) in p110δ KI DRGs (right). Each data point represents the %±SEM ( n ≥3 independent experiments). In each experiment at least 80 growth cones were counted. *p<0.02. (C) Example of DRG explants in each group, which have been stained for βIII-tubulin ( red ) and phalloidin ( green ). Scale bar, 20 µm.
Given that the absence of p110δ activity limits axonal outgrowth in embryonic neurons, we assessed the axonal growth potential of adult p110δ KI neurons. Adult peripheral nerve axons are capable of functional regeneration in mammals, with a crush injury of the sciatic nerve being a well-established injury model to study axonal growth. On day 3, extension of regenerating neuronal fibers 2 mm distal from the injury site was reduced in p110δ KI mice ( ), without any differences in the presence of cytoskeletal breakdown products 4 mm distally from the site of injury (data not shown), indicating that loss of p110δ activity does not alter normal axonal degeneration. p110δ activity has been shown to be essential for CSF-1-driven in vitro chemotaxis of macrophages,, which might affect axonal regeneration due to impaired inflammatory response at the injury site in p110δ KI mice. No changes in recruitment of macrophages (stained by F4/80) into the injured nerve were detected ( ). Next, we compared the capacity of DRG soma to upregulate growth-associated SPRR1A (small proline-rich repeat protein 1A) during regeneration. SPRR1A has been shown to peak 1-...
3 days post-injury, the sciatic nerves of WT and p110δ KI mice were fixed and cryo-sectioned. (A) Average relative fluorescence intensity profile of anti-βIII-tubulin labeling across a one-pixel line along the entire nerve segment, following cropping of the micrographs to a fixed pixel segment. (B) High-power micrographs of the sciatic nerve segments 2 mm distal to the injury, labeled with anti-βIII-tubulin (left panels and green in right panels), anti-F4/80 ( red ) and Hoechst ( blue ). Scale bar, 50 µm. (C) At 7 days post injury, L4 DRGs of WT and p110δ KI mice were fixed, vibratome-sectioned, and co-labeled with ATF3 (red) and SPRR1A (green). Percentage of SPRR1A and ATF3 co-labeling over ATF3-only positive DRG neurons in WT and p110δ KI mice. Data are from 3 WT and 4 p110δ KI mice, and are presented as mean±SEM. *p<0.05. Scale bar, 50 µm.
We next assessed whether the anatomical evidence for p110δ-dependency for optimal axonal regeneration correlated with functional recovery using an automated quantitative gait analysis system, the CatWalk, to assess recovery of locomotion following nerve injury on days 1, 3, 7, 10, 14 and 21. Prior to injury, gait analysis assessed by the CatWalk on a number of locomotor parameters revealed no differences between WT and p110δ KI mice (, ). In contrast, following unilateral injury to the sciatic nerve, p110δ KI mice showed a significant decrease in the recovery of the ability to bear weight on the injured paw ( ). Whilst both WT and p110δ KI mice display functional recovery during the first 10 days post-lesion, the relative paw pressure intensity in p110δ KI mice was significantly lower in comparison to WT mice (; p<0,001, 2-way repeated measured ANOVA). These differences were most apparent at later time-points (day 10 and day 14 post-lesion;; p<0.05, Tukey test). These results indicate that following sciatic nerve injury, the lack of functional p110δ led to a decreased ability of the axons to undergo regenerative growth, which in turn led to decr...
Paw pressure intensity during continuous locomotion was assessed using the CatWalk quantitative gait analysis system. Recovery in locomotion was analyzed on days 1, 3, 7, 10, 14 and 21 post-injury. (A) Intensity profiles of 5 steps during a single run from each paw, 7 days post-injury. Each intensity profile shows two traces, a higher trace of the maximal relative intensity and a lower trace of the average relative intensity. (B) Reduced relative paw pressure intensity for each paw during recovery in p110δ KI mice compared to WT mice. Data presented is the mean±SEM of ≥6 animals. p<0.001, 2-way repeated measures ANOVA.
p110α lz and p110δ lz mice were transferred into ice-cold PBS, and fixed for 1 h on ice in fixing buffer (4% paraformaldehyde/0.2% glutaraldehyde/2 mM MgCl 2 /5 mM EGTA/0.02% NP40). Embryos were then washed 3 times in washing buffer (PBS/2 mM MgCl 2 /0.01% sodium deoxycholate/0.02% NP40/5 mM EGTA) and post-fixed in 4% paraformaldehyde for 1 h. β-galactosidase expression was visualized by incubation in X-gal developing buffer (PBS/5 mM K 3 Fe(CN) 6 /5 mM K 4 (CN) 6 /2 mM MgCl 2 /0.01% sodium deoxycholate/0.02% NP40, 1 mg/ml X-gal) overnight at room temperature. Embryos were then washed in PBS, post-fixed in 4% paraformaldehyde for 1 h, and washed twice in distilled water before dehydration though a series of 70% ethanol, 95% ethanol, and 100% ethanol. Following incubation in methylsalicylate for 1 h, embryos were rehydrated in 100% ethanol, 95% ethanol, 70% ethanol, and 30% ethanol and finally washed in PBS before being embedded in 10% gelatin and vibratome-sectioned at 50 µm.
For in vivo regeneration studies, 6 to 8 week-old male mice were used. The mice were assessed before surgery to establish baseline-walking patterns. Animals were anesthetized with a mixture of medetomidine (0.5 mg/kg) and ketamine (75 mg/kg), and the left sciatic nerve was exposed. A crush injury was performed 10 mm distal to the obturator tendon using forceps compression (10 sec) and the crush site labeled with lamp black. The muscle and skin layers were sutured and animals were allowed to recover in the cage post-operatively.
The CatWalk gait analysis system was used to assess functional recovery of locomotion following sciatic nerve crush injury. The animal traversed a meter long walkway with a glass floor and 2 perspex walls spaced 8 cm apart, housed in a darkened room. Light from 2 encased white fluorescent tubes entered the glass floor through the distal edge of the glass, and was totally internally reflected. Light scatters only where a paw contacts the glass, illuminating the area of paw contact. This reflected light was captured using videocamera (Sentech 705, 8.5 mm, f = 1.4, variable focus and variable iris) equipped with a wide-angle objective and a frame grabber (Matrix Vision SG-board) connected to a PC running the CatWalk 500 software for capture and analysis. Each mouse ran across the CatWalk one day before surgery to establish baseline locomotor parameters. Following surgery, animals ran the Catwalk on days 1, 3, 7, 10, 14 and 21. The program was set to capture the paw prints from the middle section of the run. At least 2 runs per animal were performed on each day. Data was analyzed by labeling all areas containing one or more pixels above a certain analysis threshold. In...
Machine-readable layer
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"description": "Evidence-backed execution summary for Control of Axonal Growth and Regeneration of Sensory Neurons by the p110δ PI 3-Kinase methods from Control of Axonal Growth and Regeneration of Sensory Neurons by the p110δ PI 3-Kinase.",
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"step": [
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"name": "Inactivation of p110δ increases the vulnerability of sensory neurons to growth cone collapse and decreases axona...",
"text": "(A) Growth cone collapse in DRG explants induced by LY294002 (a pan-PI3K inhibitor) or IC87114 (a p110δ-selective inhibitor). Both drugs were used at 10 µM for 10 min ( n ≥6 independent experiments). *p<0.01. (B) E13.5 DRG explants were cultured for 24 h on 20 µg/ml laminin and treated with Sema3A (0.3 µg/ml) for 30 min. Increased growth cone collapse in response to Sema3A (0.3 µg/ml) in p110δ KI DRGs (right). Each data point represents the %±SEM ( n ≥3 independent experiments). In each experiment at least 80 growth cones were counted. *p<0.02. (C) Example of DRG explants in each group, which have been stained for βIII-tubulin ( red ) and phalloidin ( green ). Scale bar, 20 µm."
},
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"name": "Reduced axonal regeneration in the injured sciatic nerve of mice with inactive p110δ",
"text": "Given that the absence of p110δ activity limits axonal outgrowth in embryonic neurons, we assessed the axonal growth potential of adult p110δ KI neurons. Adult peripheral nerve axons are capable of functional regeneration in mammals, with a crush injury of the sciatic nerve being a well-established injury model to study axonal growth. On day 3, extension of regenerating neuronal fibers 2 mm distal from the injury site was reduced in p110δ KI mice ( ), without any differences in the presence of cytoskeletal breakdown products 4 mm distally from the site of injury (data not shown), indicating that loss of p110δ activity does not alter normal axonal degeneration. p110δ activity has been shown to be essential for CSF-1-driven in vitro chemotaxis of macrophages,, which might affect axonal regeneration due to impaired inflammatory response at the injury site in p..."
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"position": 3,
"name": "Reduced axonal regeneration in the injured sciatic nerve of mice with inactive p110δ",
"text": "3 days post-injury, the sciatic nerves of WT and p110δ KI mice were fixed and cryo-sectioned. (A) Average relative fluorescence intensity profile of anti-βIII-tubulin labeling across a one-pixel line along the entire nerve segment, following cropping of the micrographs to a fixed pixel segment. (B) High-power micrographs of the sciatic nerve segments 2 mm distal to the injury, labeled with anti-βIII-tubulin (left panels and green in right panels), anti-F4/80 ( red ) and Hoechst ( blue ). Scale bar, 50 µm. (C) At 7 days post injury, L4 DRGs of WT and p110δ KI mice were fixed, vibratome-sectioned, and co-labeled with ATF3 (red) and SPRR1A (green). Percentage of SPRR1A and ATF3 co-labeling over ATF3-only positive DRG neurons in WT and p110δ KI mice. Data are from 3 WT and 4 p110δ KI mice, and are presented as mean±SEM. *p<0.05. Scale bar, 50 µm."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Inhibition of axonal regeneration correlates with impaired functional recovery",
"text": "We next assessed whether the anatomical evidence for p110δ-dependency for optimal axonal regeneration correlated with functional recovery using an automated quantitative gait analysis system, the CatWalk, to assess recovery of locomotion following nerve injury on days 1, 3, 7, 10, 14 and 21. Prior to injury, gait analysis assessed by the CatWalk on a number of locomotor parameters revealed no differences between WT and p110δ KI mice (, ). In contrast, following unilateral injury to the sciatic nerve, p110δ KI mice showed a significant decrease in the recovery of the ability to bear weight on the injured paw ( ). Whilst both WT and p110δ KI mice display functional recovery during the first 10 days post-lesion, the relative paw pressure intensity in p110δ KI mice was significantly lower in comparison to WT mice (; p<0,001, 2-way repeated measured ANOVA). Thes..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Inhibition of axonal regeneration correlates with impaired functional recovery",
"text": "Paw pressure intensity during continuous locomotion was assessed using the CatWalk quantitative gait analysis system. Recovery in locomotion was analyzed on days 1, 3, 7, 10, 14 and 21 post-injury. (A) Intensity profiles of 5 steps during a single run from each paw, 7 days post-injury. Each intensity profile shows two traces, a higher trace of the maximal relative intensity and a lower trace of the average relative intensity. (B) Reduced relative paw pressure intensity for each paw during recovery in p110δ KI mice compared to WT mice. Data presented is the mean±SEM of ≥6 animals. p<0.001, 2-way repeated measures ANOVA."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Embryo and tissue preparation",
"text": "p110α lz and p110δ lz mice were transferred into ice-cold PBS, and fixed for 1 h on ice in fixing buffer (4% paraformaldehyde/0.2% glutaraldehyde/2 mM MgCl 2 /5 mM EGTA/0.02% NP40). Embryos were then washed 3 times in washing buffer (PBS/2 mM MgCl 2 /0.01% sodium deoxycholate/0.02% NP40/5 mM EGTA) and post-fixed in 4% paraformaldehyde for 1 h. β-galactosidase expression was visualized by incubation in X-gal developing buffer (PBS/5 mM K 3 Fe(CN) 6 /5 mM K 4 (CN) 6 /2 mM MgCl 2 /0.01% sodium deoxycholate/0.02% NP40, 1 mg/ml X-gal) overnight at room temperature. Embryos were then washed in PBS, post-fixed in 4% paraformaldehyde for 1 h, and washed twice in distilled water before dehydration though a series of 70% ethanol, 95% ethanol, and 100% ethanol. Following incubation in methylsalicylate for 1 h, embryos were rehydrated in 100% ethanol, 95% ethanol, 70% ethanol, and..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Sciatic nerve crush",
"text": "For in vivo regeneration studies, 6 to 8 week-old male mice were used. The mice were assessed before surgery to establish baseline-walking patterns. Animals were anesthetized with a mixture of medetomidine (0.5 mg/kg) and ketamine (75 mg/kg), and the left sciatic nerve was exposed. A crush injury was performed 10 mm distal to the obturator tendon using forceps compression (10 sec) and the crush site labeled with lamp black. The muscle and skin layers were sutured and animals were allowed to recover in the cage post-operatively."
},
{
"@type": "HowToStep",
"position": 8,
"name": "The CatWalk",
"text": "The CatWalk gait analysis system was used to assess functional recovery of locomotion following sciatic nerve crush injury. The animal traversed a meter long walkway with a glass floor and 2 perspex walls spaced 8 cm apart, housed in a darkened room. Light from 2 encased white fluorescent tubes entered the glass floor through the distal edge of the glass, and was totally internally reflected. Light scatters only where a paw contacts the glass, illuminating the area of paw contact. This reflected light was captured using videocamera (Sentech 705, 8.5 mm, f = 1.4, variable focus and variable iris) equipped with a wide-angle objective and a frame grabber (Matrix Vision SG-board) connected to a PC running the CatWalk 500 software for capture and analysis. Each mouse ran across the CatWalk one day before surgery to establish baseline locomotor parameters. Following surgery, a..."
}
],
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"@type": "HowToTool",
"name": "Inactivation of p110δ increases the vulnerability of sensory neurons to growth cone collapse and decreases axona..."
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"name": "Inhibition of axonal regeneration correlates with impaired functional recovery"
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{
"@type": "HowToTool",
"name": "Inhibition of axonal regeneration correlates with impaired functional recovery"
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{
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"name": "The CatWalk"
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{
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"name": "Morris water maze"
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{
"@type": "HowToTool",
"name": "Immunohistochemistry"
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{
"@type": "HowToTool",
"name": "SDS-PAGE and Western blotting"
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{
"@type": "HowToTool",
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{
"@type": "HowToSupply",
"name": "Inactivation of p110δ increases the vulnerability of sensory neurons to growth cone collapse and decreases axona..."
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{
"@type": "HowToSupply",
"name": "Inactivation of p110δ increases the vulnerability of sensory neurons to growth cone collapse and decreases axona..."
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"name": "Reduced Akt and increased RhoA/PTEN signaling in neurons with inactive p110δ"
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{
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"name": "Embryo and tissue preparation"
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{
"@type": "HowToSupply",
"name": "Immunohistochemistry"
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{
"@type": "HowToSupply",
"name": "Neuronal cultures"
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{
"@type": "HowToSupply",
"name": "Immunocytochemistry"
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{
"@type": "HowToSupply",
"name": "SDS-PAGE and Western blotting"
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"headline": "Control of Axonal Growth and Regeneration of Sensory Neurons by the p110δ PI 3-Kinase",
"datePublished": "2007",
"author": [
{
"@type": "Person",
"name": "Britta J. Eickholt"
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"name": "Evangelia A. Papakonstanti"
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"name": "Wayne Pearce"
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"name": "Michelle L. Starkey"
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"name": "Antonio Bilancio"
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"name": "Anna C. Need"
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"name": "Susan M. Hall"
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"name": "Frank P. Hamers"
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"name": "Karl P. Giese"
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"name": "Elizabeth J. Bradbury"
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"name": "Bart Vanhaesebroeck"
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"identifier": "10.1371/journal.pone.0000869"
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