Transneuronal delivery of hyper-interleukin-6 enables functional recovery after severe spinal cord injury in mice methods
Aim. Evidence-backed execution summary for Transneuronal delivery of hyper-interleukin-6 enables functional recovery after severe spinal cord injury in mice methods from Transneuronal delivery of hyper-interleukin-6 enables functional recovery after severe spinal cord injury in mice.
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Cortical AAV2-hIL-6 delivery promotes RpST regeneration
reagent used in the protocol.
- Use
- The lack of functional recovery in PTEN -/- mice suggested that improved CST regeneration was not the leading cause for the AAV2-hIL-6 mediated functional recovery. As descending serotonergic (5-HT-positive) axons of the raphespinal tract (RpST) are reportedly relevant for locomotor recovery -, we...
DHT injection
reagent used in the protocol.
- Use
- Mice received bilateral intracerebroventricular injections of 30 µg of the serotonin neurotoxin 5,7-dihydroxytryptamine (DHT, Biomol) dissolved in 0.5 µl of 0.2% ascorbic acid in saline to deplete serotonergic inputs to the lumbar spinal cord. To this end, mice were anesthetized and fixed under...
AAV2-hIL-6 confers transneuronal stimulation of serotonergic fiber regeneration
reagent used in the protocol.
- Use
- To understand the mechanism underlying the bilateral, RpST-dependent functional recovery by a one-time unilateral AAV2-hIL-6 virus application into the sensorimotor cortex, we tested the hypothesis that transduced cortical motoneurons project axons to the raphe nuclei in the brain stem and that the release of h...
Intracortical injection of pups
reagent used in the protocol.
- Use
- Postnatal (P1) PTEN floxed (C57BL/6;129/J-TgH(Pten-flox)) pups were fixed on a stereotactic frame and continuously supplied with 2% isoflurane for anesthesia via a mouthpiece. A midline incision into the skin was made to expose the skull using microscissors. Since the skulls of P1 mice are still soft, the cortex cou...
Intracortical injection of adult mice
reagent used in the protocol.
- Use
- For intracortical injections, adult PTEN floxed (C57BL/6;129/J-TgH(Pten-flox)) or adult wt (C57BL/6) mice were anesthetized by intraperitoneal injection of ketamine (120 mg/kg) and xylazine (16 mg/kg) and then placed in a stereotaxic frame. A midline incision was made over the skull to open the skin and...
Injection into the red nucleus
reagent used in the protocol.
- Use
- Adult wt mice were anesthetized by intraperitoneal ketamine (100 mg/kg) and xylazine (10 mg/kg) injection and then placed in a stereotaxic frame. A midline incision was made over the skull to reveal bregma. A micro drill with a 0.5 mm bit was used to open a 1 × 1 mm window in t...
CST tracing
reagent used in the protocol.
- Use
- To trace the axons of cortical motoneurons in PTEN floxed (C57BL/6;129/J-TgH(Pten-flox)) or wt (C57BL/6) mice, we injected the axon tracer biotinylated dextran amine (BDA, 10,000 MW, 10% solution in water, Invitrogen, D1956) into the sensorimotor cortex 2 weeks before the mice were sacrificed,,. Therefore,...
Injection into the raphe nuclei
reagent used in the protocol.
- Use
- For brainstem injections, adult Rosa26 loxP-stop-loxP-tdTomato mice were anesthetized by intraperitoneal ketamine (120 mg/kg) and xylazine (16 mg/kg) injections and then placed in a stereotactic frame. A midline incision was made over the skull to reveal bregma. A micro drill with a 0.5 mm bi...
Hyper-IL-6 promotes functional recovery
Before tissue isolation and analysis, hindlimb movement had been analyzed in all four experimental groups using open-field locomotion tests according to the Basso Mouse Scale (BMS) over the postinjury period of 8 weeks. Consistent with previous reports -, the BMS score dropped down to 0 in all animals 1 day...
- Use
- Before tissue isolation and analysis, hindlimb movement had been analyzed in all four experimental groups using open-field locomotion tests according to the Basso Mouse Scale (BMS) over the postinjury period of 8 weeks. Consistent with previous reports -, the BMS score dropped down to 0 in all animals 1 day...
AAV2-hIL-6-mediated recovery depends on the regeneration of serotonergic neurons
To investigate the relevance of RpST regeneration for functional recovery, adult mice of the same genetic background were subjected to SCC and received bilateral intracortical injections of either AAV2-hIL-6 or AAV2-GFP directly afterward (Fig. ). At week 6, when functional recovery in AAV2-hIL-6 treated mice...
- Use
- To investigate the relevance of RpST regeneration for functional recovery, adult mice of the same genetic background were subjected to SCC and received bilateral intracortical injections of either AAV2-hIL-6 or AAV2-GFP directly afterward (Fig. ). At week 6, when functional recovery in AAV2-hIL-6 treated mice...
AAV2-hIL-6-mediated recovery depends on the regeneration of serotonergic neurons
Since genetic backgrounds could potentially affect regeneration, we also tested the postinjury-applied AAV2-hIL-6 treatment in non-transgenic BL6 mice, whose low potential for functional recovery has been previously documented. Eight weeks after SCC and unilateral intracortical AAV2-hIL-6 injection, BL-6 mice also...
- Use
- Since genetic backgrounds could potentially affect regeneration, we also tested the postinjury-applied AAV2-hIL-6 treatment in non-transgenic BL6 mice, whose low potential for functional recovery has been previously documented. Eight weeks after SCC and unilateral intracortical AAV2-hIL-6 injection, BL-6 mice also...
AAV2-hIL-6 confers transneuronal stimulation of serotonergic fiber regeneration
To understand the mechanism underlying the bilateral, RpST-dependent functional recovery by a one-time unilateral AAV2-hIL-6 virus application into the sensorimotor cortex, we tested the hypothesis that transduced cortical motoneurons project axons to the raphe nuclei in the brain stem and that the release of h...
- Use
- To understand the mechanism underlying the bilateral, RpST-dependent functional recovery by a one-time unilateral AAV2-hIL-6 virus application into the sensorimotor cortex, we tested the hypothesis that transduced cortical motoneurons project axons to the raphe nuclei in the brain stem and that the release of h...
AAV2-hIL-6 confers transneuronal stimulation of serotonergic fiber regeneration
We then investigated whether raphe neurons in the brain stem, which express the hIL-6 receptor GP130, receive synaptic input from transduced cortical motoneurons. To this end, we analyzed 5-HT stained brain stem tissue from mice 8 weeks after SCC and cortical AAV2-hIL-6 treatment as described above...
- Use
- We then investigated whether raphe neurons in the brain stem, which express the hIL-6 receptor GP130, receive synaptic input from transduced cortical motoneurons. To this end, we analyzed 5-HT stained brain stem tissue from mice 8 weeks after SCC and cortical AAV2-hIL-6 treatment as described above...
DRG neuron two-compartment cultures
Dorsal root ganglion-(DRG) neurons were harvested from adult mice. Isolated DRG (T8-L5) were incubated in 0.25% trypsin/EDTA (GE Healthcare) and 0.3% collagenase Type IA (Sigma) dissolved in DMEM (Invitrogen) at 37 °C and 5% CO 2 for 45 min followed by mechanical dissociation. Cells were resuspende...
- Use
- Dorsal root ganglion-(DRG) neurons were harvested from adult mice. Isolated DRG (T8-L5) were incubated in 0.25% trypsin/EDTA (GE Healthcare) and 0.3% collagenase Type IA (Sigma) dissolved in DMEM (Invitrogen) at 37 °C and 5% CO 2 for 45 min followed by mechanical dissociation. Cells were resuspende...
Dissociated retinal cell cultures
After fixation with 4% PFA for 30 min at RT, cell cultures were processed for immunocytochemical staining with a βIII-tubulin antibody (1:2,000, BioLegend, RRID:AB_2313773). All RGCs with regenerated neurites were photographed using a fluorescence microscope ( × 200, Axio Observer.D1, Zeiss), and neuri...
- Use
- After fixation with 4% PFA for 30 min at RT, cell cultures were processed for immunocytochemical staining with a βIII-tubulin antibody (1:2,000, BioLegend, RRID:AB_2313773). All RGCs with regenerated neurites were photographed using a fluorescence microscope ( × 200, Axio Observer.D1, Zeiss), and neuri...
Immunohistochemistry
Cryosections of brains, medullas and spinal cords were thawed, washed in PBS for 15 min and permeabilized by 10 min incubation in 100% Methanol (Sigma). Sections were stained with antibodies against the HA-tag (1:500; Sigma; RRID:AB_260070), pSTAT3 (1:200; RRID: AB_2491009), pAKT-Thr308 (1:500; RRID: AB_...
- Use
- Cryosections of brains, medullas and spinal cords were thawed, washed in PBS for 15 min and permeabilized by 10 min incubation in 100% Methanol (Sigma). Sections were stained with antibodies against the HA-tag (1:500; Sigma; RRID:AB_260070), pSTAT3 (1:200; RRID: AB_2491009), pAKT-Thr308 (1:500; RRID: AB_...
Dissociated retinal cell cultures
Software used for acquisition, scoring, statistics, or reporting.
- Use
- After fixation with 4% PFA for 30 min at RT, cell cultures were processed for immunocytochemical staining with a βIII-tubulin antibody (1:2,000, BioLegend, RRID:AB_2313773). All RGCs with regenerated neurites were photographed using a fluorescence microscope ( × 200, Axio Observer.D1, Zeiss), and neuri...
Analysis and statistics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- For all analyses, including BMS testing, CST regeneration/sprouting/dieback, RpST regeneration, characterization of DHT treatment, and immunohistochemistry, individual mice or samples of respective animals were randomly labeled with different letters in a way that the investigator was blinded to the identity of coho...
Before you run
What should be confirmed before execution?
First confirmation
Equipment is listed but no product mappings are linked.
Confirm before execution
This page is backed by a publishable Replication Data Ledger package with zero critical source-verification issues.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
Hyper-IL-6 promotes CST regeneration
Next, we tested whether AAV2-hIL-6 application, PTEN -/-, or their combination affect corticospinal tract (CST) regeneration following complete spinal cord crush (SCC). As AAV2 reaches higher neuronal transduction rates in newborn animals,, and to keep the methodology comparable to these previous studies, PTEN-floxed mice received injections of either AAV2-Cre (PTEN -/- ) or AAV2-GFP (PTEN +/+ ) into the left sensorimotor cortex at postnatal day 1 (P1). After 7 weeks, mice were subjected to SCC (Fig. ). Each received a second injection of either AAV2-hIL-6 or AAV2-GFP into the left sensorimotor cortex immediately afterward (Fig. ), resulting in four experimental groups: (a) Control animals that had received AAV2-GFP injections twice (PTEN +/+ ), (b) PTEN-floxed mice that were treated with AAV2-Cre and later with AAV2-GFP (PTEN -/&...
Hyper-IL-6 promotes functional recovery
Before tissue isolation and analysis, hindlimb movement had been analyzed in all four experimental groups using open-field locomotion tests according to the Basso Mouse Scale (BMS) over the postinjury period of 8 weeks. Consistent with previous reports -, the BMS score dropped down to 0 in all animals 1 day after injury, also indicating the completeness of the SCC (Fig. and Fig. ). Over time, control animals developed only active ankle movements, including spasms (Supplementary video ),, resulting in an average final score of 2 (Fig. and Fig. ). Despite the positive effect on CST-regeneration (Fig. ), PTEN -/- alone did not significantly improve the BMS score compared to AAV2-GFP treated controls (Fig. and Fig.; Supplementary video ). However, AAV2-hIL-6 treatment increased the BMS score to ∼4 by restor...
Cortical AAV2-hIL-6 delivery promotes RpST regeneration
The lack of functional recovery in PTEN -/- mice suggested that improved CST regeneration was not the leading cause for the AAV2-hIL-6 mediated functional recovery. As descending serotonergic (5-HT-positive) axons of the raphespinal tract (RpST) are reportedly relevant for locomotor recovery -, we stained and analyzed these axons in sagittal spinal cord sections of the same animals used in the previous experiments (Figs., ). Control (AAV2-GFP) and PTEN -/- mice only revealed sprouting of 5-HT-positive axons over distances of less than 1 mm beyond the crush site (Fig. and Fig. ). Remarkably, AAV2-hIL-6 treated mice showed more, and longer-distance regeneration of serotonergic fibers than controls or PTEN -/- (Fig. and Fig. ), with the longest axons reaching distances of >7 mm. Combinatorial treat...
AAV2-hIL-6-mediated recovery depends on the regeneration of serotonergic neurons
To investigate the relevance of RpST regeneration for functional recovery, adult mice of the same genetic background were subjected to SCC and received bilateral intracortical injections of either AAV2-hIL-6 or AAV2-GFP directly afterward (Fig. ). At week 6, when functional recovery in AAV2-hIL-6 treated mice had already reached maximal levels, the neurotoxin 5,7-dihydroxytryptamine (DHT) was intracerebroventricularly injected to selectively kill serotonergic neurons (Fig. ),,. The toxin almost completely abolished 5-HT positive neurons/axons in the medulla/spinal cord as soon as 1 day after injection (Fig. ). As described previously,,, other neurons remained unaffected, indicated by the successful BDA tracing of cortical motor neurons (Fig. ), presence of other neuN positive neurons next to depleted Raphe neurons in the medulla (Fig. ), and the alm...
AAV2-hIL-6 confers transneuronal stimulation of serotonergic fiber regeneration
We then investigated whether raphe neurons in the brain stem, which express the hIL-6 receptor GP130, receive synaptic input from transduced cortical motoneurons. To this end, we analyzed 5-HT stained brain stem tissue from mice 8 weeks after SCC and cortical AAV2-hIL-6 treatment as described above (Fig. ). GFP-positive sprouts of cortical motoneurons were located near 5-HT-positive raphe neurons in cross-sections of the medulla (Fig. ). The GFP signal and hIL-6 in the pyramidal axons were detected via high-resolution transversal scans after tissue clearing (Fig. ). Western blot analyses using brain stem lysates isolated 3 weeks after intracortical viral application showed pSTAT3 signals only in samples from AAV2-hIL-6-, but not AAV2-GFP-treated animals (Fig. and Fig. ), while total STAT3 protein expression or phosphorylation of eith...
AAV2-hIL-6 confers transneuronal stimulation of serotonergic fiber regeneration
To verify that cortical motoneurons were synaptically connected with raphe neurons, we used AAV1, which, in contrast to AAV2, can trans-synaptically transduce supraspinal target neurons. We injected AAV1-Cre into the left sensorimotor cortex of Rosa26-tdTomato (RFP) reporter mice and isolated their brain stem tissue after 2 weeks. RFP-positive (transduced) serotonergic neurons were detected in both sides of the nucleus raphe pallidus (NRPa) and the nucleus raphe magnus (NRM) (Fig. ) along their entire rostrocaudal length. Additionally, we performed a complementary experiment by injecting AAV1 directly into the raphe nuclei. As AAV1 is also retrogradely transported,, we found transduced neurons in the motor cortex layer V, confirming the synaptic connection to cortical neurons (Fig. ). Thus, cortically applied AAV2-hIL-6 transneuronally activates regenerative signal...
Methods
Male and female mice were used for all experiments. Wild-type C57BL/6 and 129/Ola mice were crossbred with PTEN f/f mice (C57BL/6;129) to obtain C57BL/6;129/J-TgH (Pten-flox) animals without further crossbreeding. C57BL/6J mice were obtained from Janvier Labs. Rosa26 loxP-stop-loxP-tdTomato (Rosa-tdTomato) mice were obtained from Jackson Laboratories (Stock No: 007914). All animals were housed under the same conditions for at least ten days before the start of experiments and generally maintained at 24 °C ambient temperature and 60% humidity with a 12 h light/dark cycle and ad libitum access to food and water. All experimental procedures were approved by the local animal care committee (LANUV Recklinghausen) and conducted in compliance with federal and state guidelines for animal experiments in Germany.
Intracortical injection of pups
Postnatal (P1) PTEN floxed (C57BL/6;129/J-TgH(Pten-flox)) pups were fixed on a stereotactic frame and continuously supplied with 2% isoflurane for anesthesia via a mouthpiece. A midline incision into the skin was made to expose the skull using microscissors. Since the skulls of P1 mice are still soft, the cortex could be accessed using a 30-gauge needle to create two small holes in the left skull hemisphere with the following coordinates: -0.2 mm and 0.3 mm anteroposterior, 1.0 mm lateral to bregma. For AAV2-GFP or AAV2-Cre application, 770 nl of the virus suspensions were injected at a depth of 0.5 mm into the two sites using a pulled glass pipette connected to a nanoliter injector (Nanoject II, Drummond). To inject 770 nl, we applied 11 pulses of 70 nl at a rate of 23 nl/s and waited for 10 s after each pulse to allow distr...
Measurement outputs
What raw and processed outputs should exist?
Next, we tested whether AAV2-hIL-6 application, PTEN -/-, or their combination affect corticospinal tract (CST) regeneration following complete spinal cord cru...
- 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
Before tissue isolation and analysis, hindlimb movement had been analyzed in all four experimental groups using open-field locomotion tests according to the Basso Mouse Scale (B...
- 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
The lack of functional recovery in PTEN -/- mice suggested that improved CST regeneration was not the leading cause for the AAV2-hIL-6 mediated functional recovery....
- 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 investigate the relevance of RpST regeneration for functional recovery, adult mice of the same genetic background were subjected to SCC and received bilateral intracortical i...
- 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
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Next, we tested whether AAV2-hIL-6 application, PTEN -/-, or their combination affect corticospinal tract (CST) regeneration following complete spinal cord crush (SCC).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Next, we tested whether AAV2-hIL-6 application, PTEN -/-, or their combination affect corticospinal tract (CST) regeneration following complete spinal cord cru...; Before tissue isolation and analysis, hindlimb movement had been analyzed in all four experimental groups using open-field locomotion tests according to the Basso Mouse Scale (B...; The lack of functional recovery in PTEN -/- mice suggested that improved CST regeneration was not the leading cause for the AAV2-hIL-6 mediated functional recovery....; To investigate the relevance of RpST regeneration for functional recovery, adult mice of the same genetic background were subjected to SCC and received bilateral intracortical i....
from paperStatistical comparison
Next, we tested whether AAV2-hIL-6 application, PTEN -/-, or their combination affect corticospinal tract (CST) regeneration following complete spinal cord cru...; Before tissue isolation and analysis, hindlimb movement had been analyzed in all four experimental groups using open-field locomotion tests according to the Basso Mouse Scale (B...; The lack of functional recovery in PTEN -/- mice suggested that improved CST regeneration was not the leading cause for the AAV2-hIL-6 mediated functional recovery....; To investigate the relevance of RpST regeneration for functional recovery, adult mice of the same genetic background were subjected to SCC and received bilateral intracortical i...
from paperReporting output
Report representative outputs alongside summary comparisons for Next, we tested whether AAV2-hIL-6 application, PTEN -/-, or their combination affect corticospinal tract (CST) regeneration following complete spinal cord cru..., Before tissue isolation and analysis, hindlimb movement had been analyzed in all four experimental groups using open-field locomotion tests according to the Basso Mouse Scale (B..., The lack of functional recovery in PTEN -/- mice suggested that improved CST regeneration was not the leading cause for the AAV2-hIL-6 mediated functional recovery...., To investigate the relevance of RpST regeneration for functional recovery, adult mice of the same genetic background were subjected to SCC and received bilateral intracortical i....
inferred from protocolStructured statistical methods
Next, we tested whether AAV2-hIL-6 application, PTEN -/-, or their combination affect corticospinal tract (CST) regeneration following complete spinal cord cru...; Before tissue isolation and analysis, hindlimb movement had been analyzed in all four experimental groups using open-field locomotion tests according to the Basso Mouse Scale (B...; The lack of functional recovery in PTEN -/- mice suggested that improved CST regeneration was not the leading cause for the AAV2-hIL-6 mediated functional recovery....; To investigate the relevance of RpST regeneration for functional recovery, adult mice of the same genetic background were subjected to SCC and received bilateral intracortical i...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Next, we tested whether AAV2-hIL-6 application, PTEN -/-, or their combination affect corticospinal tract (CST) regeneration following complete spinal cord crush (SCC). As AAV2 reaches higher neuronal transduction rates in newborn animals,, and to keep the methodology comparable to these previous studies, PTEN-floxed mice received injections of either AAV2-Cre (PTEN -/- ) or AAV2-GFP (PTEN +/+ ) into the left sensorimotor cortex at postnatal day 1 (P1). After 7 weeks, mice were subjected to SCC (Fig. ). Each received a second injection of either AAV2-hIL-6 or AAV2-GFP into the left sensorimotor cortex immediately afterward (Fig. ), resulting in four experimental groups: (a) Control animals that had received AAV2-GFP injections twice (PTEN +/+ ), (b) PTEN-floxed mice that were treated with AAV2-Cre and later with AAV2-GFP (PTEN -/- ), (c) mice that received AAV2-GFP and later, after SCC, AAV2-hIL-6 (hIL-6) and (d) mice that were treated with AAV2-Cre first and, after SCC, with AAV2-hIL-6 (PTEN -/- /hIL-6). Axonal biotinylated dextran amine (BDA)-tracing of the right CST (Fig. ) was performed 6 wee...
Before tissue isolation and analysis, hindlimb movement had been analyzed in all four experimental groups using open-field locomotion tests according to the Basso Mouse Scale (BMS) over the postinjury period of 8 weeks. Consistent with previous reports -, the BMS score dropped down to 0 in all animals 1 day after injury, also indicating the completeness of the SCC (Fig. and Fig. ). Over time, control animals developed only active ankle movements, including spasms (Supplementary video ),, resulting in an average final score of 2 (Fig. and Fig. ). Despite the positive effect on CST-regeneration (Fig. ), PTEN -/- alone did not significantly improve the BMS score compared to AAV2-GFP treated controls (Fig. and Fig.; Supplementary video ). However, AAV2-hIL-6 treatment increased the BMS score to ∼4 by restoring plantar stepping with full hindlimb weight support, followed by lift-off, forward limb advancement, and reestablishment of weight support at initial contact in most of the animals (Fig. and Fig.; Supplementary video ). Combinatorial treatment (PTEN -/- +R...
The lack of functional recovery in PTEN -/- mice suggested that improved CST regeneration was not the leading cause for the AAV2-hIL-6 mediated functional recovery. As descending serotonergic (5-HT-positive) axons of the raphespinal tract (RpST) are reportedly relevant for locomotor recovery -, we stained and analyzed these axons in sagittal spinal cord sections of the same animals used in the previous experiments (Figs., ). Control (AAV2-GFP) and PTEN -/- mice only revealed sprouting of 5-HT-positive axons over distances of less than 1 mm beyond the crush site (Fig. and Fig. ). Remarkably, AAV2-hIL-6 treated mice showed more, and longer-distance regeneration of serotonergic fibers than controls or PTEN -/- (Fig. and Fig. ), with the longest axons reaching distances of >7 mm. Combinatorial treatment of AAV2-hIL-6 and PTEN -/- had no additional effect (Fig. and Fig. ). Furthermore, as measured during functional recovery analysis (Fig. ), bilateral AAV2-hIL-6 treatment did not increase RpST regeneration further compared to a unilateral application of the virus (...
To investigate the relevance of RpST regeneration for functional recovery, adult mice of the same genetic background were subjected to SCC and received bilateral intracortical injections of either AAV2-hIL-6 or AAV2-GFP directly afterward (Fig. ). At week 6, when functional recovery in AAV2-hIL-6 treated mice had already reached maximal levels, the neurotoxin 5,7-dihydroxytryptamine (DHT) was intracerebroventricularly injected to selectively kill serotonergic neurons (Fig. ),,. The toxin almost completely abolished 5-HT positive neurons/axons in the medulla/spinal cord as soon as 1 day after injection (Fig. ). As described previously,,, other neurons remained unaffected, indicated by the successful BDA tracing of cortical motor neurons (Fig. ), presence of other neuN positive neurons next to depleted Raphe neurons in the medulla (Fig. ), and the almost normal open field movement in uninjured mice evaluated 1 week after DHT application (Fig. ). However, in injured animals, 1 day after DHT application, the BMS of AAV2-hIL-6-treated mice dropped down to similar levels as in AAV-GFP treated control animals, which themselves remained unaffect...
We then investigated whether raphe neurons in the brain stem, which express the hIL-6 receptor GP130, receive synaptic input from transduced cortical motoneurons. To this end, we analyzed 5-HT stained brain stem tissue from mice 8 weeks after SCC and cortical AAV2-hIL-6 treatment as described above (Fig. ). GFP-positive sprouts of cortical motoneurons were located near 5-HT-positive raphe neurons in cross-sections of the medulla (Fig. ). The GFP signal and hIL-6 in the pyramidal axons were detected via high-resolution transversal scans after tissue clearing (Fig. ). Western blot analyses using brain stem lysates isolated 3 weeks after intracortical viral application showed pSTAT3 signals only in samples from AAV2-hIL-6-, but not AAV2-GFP-treated animals (Fig. and Fig. ), while total STAT3 protein expression or phosphorylation of either AKT or S6 remained unchanged (Fig. and Figs., ). Additionally, transverse scans through cleared brain stem tissue (Fig., Supplementary video ) from hIL-6 treated animals, and immunostaining of coronal sections showed, on average, 46% ± 4.46 SEM of...
To verify that cortical motoneurons were synaptically connected with raphe neurons, we used AAV1, which, in contrast to AAV2, can trans-synaptically transduce supraspinal target neurons. We injected AAV1-Cre into the left sensorimotor cortex of Rosa26-tdTomato (RFP) reporter mice and isolated their brain stem tissue after 2 weeks. RFP-positive (transduced) serotonergic neurons were detected in both sides of the nucleus raphe pallidus (NRPa) and the nucleus raphe magnus (NRM) (Fig. ) along their entire rostrocaudal length. Additionally, we performed a complementary experiment by injecting AAV1 directly into the raphe nuclei. As AAV1 is also retrogradely transported,, we found transduced neurons in the motor cortex layer V, confirming the synaptic connection to cortical neurons (Fig. ). Thus, cortically applied AAV2-hIL-6 transneuronally activates regenerative signaling pathways in ipsi- and contralateral raphe neurons by releasing the protein at the axon terminals. Fig. 7 CST axon collaterals project to raphe nuclei. a Schematic illustration, indicating the cortical application, anterograde axonal transport, and terminal release of AAV1-Cre into raphe nuclei (...
Male and female mice were used for all experiments. Wild-type C57BL/6 and 129/Ola mice were crossbred with PTEN f/f mice (C57BL/6;129) to obtain C57BL/6;129/J-TgH (Pten-flox) animals without further crossbreeding. C57BL/6J mice were obtained from Janvier Labs. Rosa26 loxP-stop-loxP-tdTomato (Rosa-tdTomato) mice were obtained from Jackson Laboratories (Stock No: 007914). All animals were housed under the same conditions for at least ten days before the start of experiments and generally maintained at 24 °C ambient temperature and 60% humidity with a 12 h light/dark cycle and ad libitum access to food and water. All experimental procedures were approved by the local animal care committee (LANUV Recklinghausen) and conducted in compliance with federal and state guidelines for animal experiments in Germany.
Postnatal (P1) PTEN floxed (C57BL/6;129/J-TgH(Pten-flox)) pups were fixed on a stereotactic frame and continuously supplied with 2% isoflurane for anesthesia via a mouthpiece. A midline incision into the skin was made to expose the skull using microscissors. Since the skulls of P1 mice are still soft, the cortex could be accessed using a 30-gauge needle to create two small holes in the left skull hemisphere with the following coordinates: -0.2 mm and 0.3 mm anteroposterior, 1.0 mm lateral to bregma. For AAV2-GFP or AAV2-Cre application, 770 nl of the virus suspensions were injected at a depth of 0.5 mm into the two sites using a pulled glass pipette connected to a nanoliter injector (Nanoject II, Drummond). To inject 770 nl, we applied 11 pulses of 70 nl at a rate of 23 nl/s and waited for 10 s after each pulse to allow distribution of the virus solution. After injection, the pipette was left in place for 1 min before being carefully withdrawn, and the incision carefully closed with a 4-0 black silk suture.
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Transneuronal delivery of hyper-interleukin-6 enables functional recovery after severe spinal cord injury in mice methods",
"description": "Evidence-backed execution summary for Transneuronal delivery of hyper-interleukin-6 enables functional recovery after severe spinal cord injury in mice methods from Transneuronal delivery of hyper-interleukin-6 enables functional recovery after severe spinal cord injury in mice.",
"totalTime": "PT79680M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Hyper-IL-6 promotes CST regeneration",
"text": "Next, we tested whether AAV2-hIL-6 application, PTEN -/-, or their combination affect corticospinal tract (CST) regeneration following complete spinal cord crush (SCC). As AAV2 reaches higher neuronal transduction rates in newborn animals,, and to keep the methodology comparable to these previous studies, PTEN-floxed mice received injections of either AAV2-Cre (PTEN -/- ) or AAV2-GFP (PTEN +/+ ) into the left sensorimotor cortex at postnatal day 1 (P1). After 7 weeks, mice were subjected to SCC (Fig. ). Each received a second injection of either AAV2-hIL-6 or AAV2-GFP into the left sensorimotor cortex immediately afterward (Fig. ), resulting in four experimental groups: (a) Control animals that had received AAV2-GFP injections twice (PTEN +/+ ), (b) PTEN-floxed mice that were treated with AAV2-Cre and later with AAV2-GFP (PTEN -/&..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Hyper-IL-6 promotes functional recovery",
"text": "Before tissue isolation and analysis, hindlimb movement had been analyzed in all four experimental groups using open-field locomotion tests according to the Basso Mouse Scale (BMS) over the postinjury period of 8 weeks. Consistent with previous reports -, the BMS score dropped down to 0 in all animals 1 day after injury, also indicating the completeness of the SCC (Fig. and Fig. ). Over time, control animals developed only active ankle movements, including spasms (Supplementary video ),, resulting in an average final score of 2 (Fig. and Fig. ). Despite the positive effect on CST-regeneration (Fig. ), PTEN -/- alone did not significantly improve the BMS score compared to AAV2-GFP treated controls (Fig. and Fig.; Supplementary video ). However, AAV2-hIL-6 treatment increased the BMS score to ∼4 by restor..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Cortical AAV2-hIL-6 delivery promotes RpST regeneration",
"text": "The lack of functional recovery in PTEN -/- mice suggested that improved CST regeneration was not the leading cause for the AAV2-hIL-6 mediated functional recovery. As descending serotonergic (5-HT-positive) axons of the raphespinal tract (RpST) are reportedly relevant for locomotor recovery -, we stained and analyzed these axons in sagittal spinal cord sections of the same animals used in the previous experiments (Figs., ). Control (AAV2-GFP) and PTEN -/- mice only revealed sprouting of 5-HT-positive axons over distances of less than 1 mm beyond the crush site (Fig. and Fig. ). Remarkably, AAV2-hIL-6 treated mice showed more, and longer-distance regeneration of serotonergic fibers than controls or PTEN -/- (Fig. and Fig. ), with the longest axons reaching distances of >7 mm. Combinatorial treat..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "AAV2-hIL-6-mediated recovery depends on the regeneration of serotonergic neurons",
"text": "To investigate the relevance of RpST regeneration for functional recovery, adult mice of the same genetic background were subjected to SCC and received bilateral intracortical injections of either AAV2-hIL-6 or AAV2-GFP directly afterward (Fig. ). At week 6, when functional recovery in AAV2-hIL-6 treated mice had already reached maximal levels, the neurotoxin 5,7-dihydroxytryptamine (DHT) was intracerebroventricularly injected to selectively kill serotonergic neurons (Fig. ),,. The toxin almost completely abolished 5-HT positive neurons/axons in the medulla/spinal cord as soon as 1 day after injection (Fig. ). As described previously,,, other neurons remained unaffected, indicated by the successful BDA tracing of cortical motor neurons (Fig. ), presence of other neuN positive neurons next to depleted Raphe neurons in the medulla (Fig. ), and the alm..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "AAV2-hIL-6 confers transneuronal stimulation of serotonergic fiber regeneration",
"text": "We then investigated whether raphe neurons in the brain stem, which express the hIL-6 receptor GP130, receive synaptic input from transduced cortical motoneurons. To this end, we analyzed 5-HT stained brain stem tissue from mice 8 weeks after SCC and cortical AAV2-hIL-6 treatment as described above (Fig. ). GFP-positive sprouts of cortical motoneurons were located near 5-HT-positive raphe neurons in cross-sections of the medulla (Fig. ). The GFP signal and hIL-6 in the pyramidal axons were detected via high-resolution transversal scans after tissue clearing (Fig. ). Western blot analyses using brain stem lysates isolated 3 weeks after intracortical viral application showed pSTAT3 signals only in samples from AAV2-hIL-6-, but not AAV2-GFP-treated animals (Fig. and Fig. ), while total STAT3 protein expression or phosphorylation of eith..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "AAV2-hIL-6 confers transneuronal stimulation of serotonergic fiber regeneration",
"text": "To verify that cortical motoneurons were synaptically connected with raphe neurons, we used AAV1, which, in contrast to AAV2, can trans-synaptically transduce supraspinal target neurons. We injected AAV1-Cre into the left sensorimotor cortex of Rosa26-tdTomato (RFP) reporter mice and isolated their brain stem tissue after 2 weeks. RFP-positive (transduced) serotonergic neurons were detected in both sides of the nucleus raphe pallidus (NRPa) and the nucleus raphe magnus (NRM) (Fig. ) along their entire rostrocaudal length. Additionally, we performed a complementary experiment by injecting AAV1 directly into the raphe nuclei. As AAV1 is also retrogradely transported,, we found transduced neurons in the motor cortex layer V, confirming the synaptic connection to cortical neurons (Fig. ). Thus, cortically applied AAV2-hIL-6 transneuronally activates regenerative signal..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Methods",
"text": "Male and female mice were used for all experiments. Wild-type C57BL/6 and 129/Ola mice were crossbred with PTEN f/f mice (C57BL/6;129) to obtain C57BL/6;129/J-TgH (Pten-flox) animals without further crossbreeding. C57BL/6J mice were obtained from Janvier Labs. Rosa26 loxP-stop-loxP-tdTomato (Rosa-tdTomato) mice were obtained from Jackson Laboratories (Stock No: 007914). All animals were housed under the same conditions for at least ten days before the start of experiments and generally maintained at 24 °C ambient temperature and 60% humidity with a 12 h light/dark cycle and ad libitum access to food and water. All experimental procedures were approved by the local animal care committee (LANUV Recklinghausen) and conducted in compliance with federal and state guidelines for animal experiments in Germany."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Intracortical injection of pups",
"text": "Postnatal (P1) PTEN floxed (C57BL/6;129/J-TgH(Pten-flox)) pups were fixed on a stereotactic frame and continuously supplied with 2% isoflurane for anesthesia via a mouthpiece. A midline incision into the skin was made to expose the skull using microscissors. Since the skulls of P1 mice are still soft, the cortex could be accessed using a 30-gauge needle to create two small holes in the left skull hemisphere with the following coordinates: -0.2 mm and 0.3 mm anteroposterior, 1.0 mm lateral to bregma. For AAV2-GFP or AAV2-Cre application, 770 nl of the virus suspensions were injected at a depth of 0.5 mm into the two sites using a pulled glass pipette connected to a nanoliter injector (Nanoject II, Drummond). To inject 770 nl, we applied 11 pulses of 70 nl at a rate of 23 nl/s and waited for 10 s after each pulse to allow distr..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Hyper-IL-6 promotes functional recovery"
},
{
"@type": "HowToTool",
"name": "AAV2-hIL-6-mediated recovery depends on the regeneration of serotonergic neurons"
},
{
"@type": "HowToTool",
"name": "AAV2-hIL-6-mediated recovery depends on the regeneration of serotonergic neurons"
},
{
"@type": "HowToTool",
"name": "AAV2-hIL-6 confers transneuronal stimulation of serotonergic fiber regeneration"
},
{
"@type": "HowToTool",
"name": "AAV2-hIL-6 confers transneuronal stimulation of serotonergic fiber regeneration"
},
{
"@type": "HowToTool",
"name": "DRG neuron two-compartment cultures"
},
{
"@type": "HowToTool",
"name": "Dissociated retinal cell cultures"
},
{
"@type": "HowToTool",
"name": "Immunohistochemistry"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Cortical AAV2-hIL-6 delivery promotes RpST regeneration"
},
{
"@type": "HowToSupply",
"name": "DHT injection"
},
{
"@type": "HowToSupply",
"name": "AAV2-hIL-6 confers transneuronal stimulation of serotonergic fiber regeneration"
},
{
"@type": "HowToSupply",
"name": "Intracortical injection of pups"
},
{
"@type": "HowToSupply",
"name": "Intracortical injection of adult mice"
},
{
"@type": "HowToSupply",
"name": "Injection into the red nucleus"
},
{
"@type": "HowToSupply",
"name": "CST tracing"
},
{
"@type": "HowToSupply",
"name": "Injection into the raphe nuclei"
}
],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Transneuronal delivery of hyper-interleukin-6 enables functional recovery after severe spinal cord injury in mice",
"datePublished": "2021",
"author": [
{
"@type": "Person",
"name": "Marco Leibinger"
},
{
"@type": "Person",
"name": "Charlotte Zeitler"
},
{
"@type": "Person",
"name": "Philipp Gobrecht"
},
{
"@type": "Person",
"name": "Anastasia Andreadaki"
},
{
"@type": "Person",
"name": "Günter Gisselmann"
},
{
"@type": "Person",
"name": "Dietmar Fischer"
}
],
"identifier": "10.1038/s41467-020-20112-4"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Transneuronal delivery of hyper-interleukin-6 enables functional recovery after severe spinal cord injury in mice methods",
"item": "https://replicatescience.com/experiments/transneuronal-delivery-of-hyper-interleukin-6-enables-functional-recovery-after-severe-spinal-cord-injury-in-mice-methods-marco-leibinger-pmc7810685/transneuronal-delivery-of-hyper-interleukin-6-enables-functional-recovery-after-severe-spinal-cord-i-mlph8v8j"
}
]
}
]