Insulin-like growth factor 1 deficiency exacerbates hypertension-induced cerebral microhemorrhages in mice, mimicking the aging phenotype methods
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Measurement of serum IGF-1 levels
Submandibular venous blood was collected into microcentrifuge tubes using a sterile lancet (Medipoint, Mineola, NY, USA) according to the manufacturer's instructions. Whole blood was centrifuged at 2500 g for 20 min at 4 °C to collect serum, which was then stored at -80 °C. IGF-1 concentration in the serum samples was measured by ELISA (R&D Systems, Minneapolis, MN, USA) as reported (Toth et al., ). An IGF-1 control sample, with aliquots stored at -80 °C, was included on each plate. Serum IGF-1 levels are reported in ng mL -1.
Analysis of gait function
To assess gait function and the spatial and temporal aspects of interlimb coordination, the animals were tested using the CatWalk System (Noldus Information Technology Inc., Leesburg, VA, USA), as reported (Toth et al., ). Briefly, animals were trained to cross the walkway and then, in a dark room, had five consecutive volunteer runs on the day before and on each day after induction of hypertension. CMH-related changes in speed, base of support, interpaw phase dispersion/phase lag, stride length, and regularity index were assessed. The regularity index (%) is a fractional measure of interpaw coordination, which expresses the number of normal step sequence patterns relative to the total number of paw placements. Its value in healthy animals is ~100%. Phase dispersion is a measure of the temporal relationship between placement of two hind paws within a step cycle.
Histological analysis of intracerebral hemorrhages
Mice were euthanized and transcardially perfused with ice-cold heparinized PBS for 5 min and subsequently decapitated as reported (Toth et al., ). Then, the brains were isolated and fixed in 10% formalin at room temperature for one day. The next day, the brains were placed in fresh 10% formalin (at 4 °C, for 2 days), then in 70% ethanol (at 4 °C, for 2 days), followed by embedding in paraffin. The brains were serially sectioned at 8 µm thickness, yielding approximately 1500 sections per brain. The first two sections of every five section were stained with hematoxylin to reveal the brain structure and diaminobenzidine (DAB) to highlight the presence of hemorrhages. DAB turns into dark brown when it undergoes a reaction with peroxidases present in red blood cells, therefore allowing precise detection of extravasated blood cells in...
Assessment of hypertension-induced MMP activation in the cerebral vessels in situ
Mice from each experimental group were temporarily anesthetized with ketamine/xylazine and injected retro-orbitally with a 100 µL dose of 40 nmol L -1 MMPsense 645 FAST substrate (PerkinElmer Inc., Boston, MA, USA; 3 µmol L -1; at 37 °C, for 6 h, in the dark), as reported (Toth et al., ). This substrate is normally optically inert. Once it is cleaved, its subunits become excitable at 649 nm and emit a red signal that can be measured as an indicator of activity of MMP 2, 3, 7, 9, 12, and 13. After 12 h of circulation of the substrate, animals were transcardially perfused with ice-cold PBS containing 1 × heparin and FITC-dextran (to highlight the vascular lumen). Then, the mice were decapitated and the brains were removed and cut in half. From the left hemisphere, the frontal...
Assessment of hypertension-induced hypertrophy of penetrating arterioles
To determine the effect of IGF-1 deficiency on adaptive hypertension-induced hypertrophy of the arteriolar wall, on day 10 postinduction of hypertension, brains were perfusion-fixed (4% ice-cold paraformaldehyde; at 100 mmHg). Then, frozen OCT-embedded sagittal sections (35 µm) were cut and stored free-floating in cryoprotectant solution (25% glycerol, 25% ethylene glycol, 25% of 0.1 m phosphate buffer, and 25% water) at -20 °C. Sections were rinsed with Tris-buffered saline (TBS), permeabilized with TBS with 0.05% Tween-20. Antigen retrieval was achieved using 10 m m citrate buffer (10 m m sodium citrate and 0.05% Tween 20, pH 6.0) at 90 °C for 20 min followed by 1% sodium borohydride in PBS at room temperature for 30 min. After blocking with 5% BSA and 1% fish gelatin i...
Determination of expression changes in CMH-related genes by quantitative real-time RT-PCR
To predict gene targets associated with vascular fragility and CMHs, we used the IRIDESCENT (Wren & Garner, ) text mining package, as described (Toth et al.,, ). Using this method, important genes relevant for the pathogenesis of CMHs and structural integrity of the vasculature were identified (Table ). The mRNA expression of these genes in cerebral arteries isolated on day 10 post-induction of hypertension was analyzed by a quantitative real-time RT-PCR technique using a Strategen MX3000 platform, as previously reported (Toth et al., ). In brief, total RNA was isolated with a Mini RNA Isolation Kit (Zymo Research, Orange, CA, USA) and was reverse-transcribed using Superscript III RT (Invitrogen). Amplification efficiencies were determined using a dilution series of a standard vascular sample. Quantification was performed using the efficiency...
Measurement outputs
What raw and processed outputs should exist?
Gait recovery and locomotor function
- 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
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Acquisition
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inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary readouts for this experiment: Gait recovery and locomotor function.
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inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function.
inferred from protocolSource and audit
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Evidence quotes (6)
Submandibular venous blood was collected into microcentrifuge tubes using a sterile lancet (Medipoint, Mineola, NY, USA) according to the manufacturer's instructions. Whole blood was centrifuged at 2500 g for 20 min at 4 °C to collect serum, which was then stored at -80 °C. IGF-1 concentration in the serum samples was measured by ELISA (R&D Systems, Minneapolis, MN, USA) as reported (Toth et al., ). An IGF-1 control sample, with aliquots stored at -80 °C, was included on each plate. Serum IGF-1 levels are reported in ng mL -1.
To assess gait function and the spatial and temporal aspects of interlimb coordination, the animals were tested using the CatWalk System (Noldus Information Technology Inc., Leesburg, VA, USA), as reported (Toth et al., ). Briefly, animals were trained to cross the walkway and then, in a dark room, had five consecutive volunteer runs on the day before and on each day after induction of hypertension. CMH-related changes in speed, base of support, interpaw phase dispersion/phase lag, stride length, and regularity index were assessed. The regularity index (%) is a fractional measure of interpaw coordination, which expresses the number of normal step sequence patterns relative to the total number of paw placements. Its value in healthy animals is ~100%. Phase dispersion is a measure of the temporal relationship between placement of two hind paws within a step cycle.
Mice were euthanized and transcardially perfused with ice-cold heparinized PBS for 5 min and subsequently decapitated as reported (Toth et al., ). Then, the brains were isolated and fixed in 10% formalin at room temperature for one day. The next day, the brains were placed in fresh 10% formalin (at 4 °C, for 2 days), then in 70% ethanol (at 4 °C, for 2 days), followed by embedding in paraffin. The brains were serially sectioned at 8 µm thickness, yielding approximately 1500 sections per brain. The first two sections of every five section were stained with hematoxylin to reveal the brain structure and diaminobenzidine (DAB) to highlight the presence of hemorrhages. DAB turns into dark brown when it undergoes a reaction with peroxidases present in red blood cells, therefore allowing precise detection of extravasated blood cells in the parenchyma of the brain. All stained sections were screened by a reader blinded to the treatment groups, and images were acquired in the evidence of a positive DAB reaction. Digital images were analyzed with imagej software (NIH) to identify the location and quantify the number and size of hemo...
Mice from each experimental group were temporarily anesthetized with ketamine/xylazine and injected retro-orbitally with a 100 µL dose of 40 nmol L -1 MMPsense 645 FAST substrate (PerkinElmer Inc., Boston, MA, USA; 3 µmol L -1; at 37 °C, for 6 h, in the dark), as reported (Toth et al., ). This substrate is normally optically inert. Once it is cleaved, its subunits become excitable at 649 nm and emit a red signal that can be measured as an indicator of activity of MMP 2, 3, 7, 9, 12, and 13. After 12 h of circulation of the substrate, animals were transcardially perfused with ice-cold PBS containing 1 × heparin and FITC-dextran (to highlight the vascular lumen). Then, the mice were decapitated and the brains were removed and cut in half. From the left hemisphere, the frontal cortex containing the photoactive substrate was isolated and homogenized. To quantify MMP activity, the background-corrected fluorescence (Ex: 649 nm, Em: 666 nm) was measured spectrophotofluorometrically using a microplate reader and normalized to tissue weight, as described (Toth e...
To determine the effect of IGF-1 deficiency on adaptive hypertension-induced hypertrophy of the arteriolar wall, on day 10 postinduction of hypertension, brains were perfusion-fixed (4% ice-cold paraformaldehyde; at 100 mmHg). Then, frozen OCT-embedded sagittal sections (35 µm) were cut and stored free-floating in cryoprotectant solution (25% glycerol, 25% ethylene glycol, 25% of 0.1 m phosphate buffer, and 25% water) at -20 °C. Sections were rinsed with Tris-buffered saline (TBS), permeabilized with TBS with 0.05% Tween-20. Antigen retrieval was achieved using 10 m m citrate buffer (10 m m sodium citrate and 0.05% Tween 20, pH 6.0) at 90 °C for 20 min followed by 1% sodium borohydride in PBS at room temperature for 30 min. After blocking with 5% BSA and 1% fish gelatin in TBS at room temperature for 2 h, sections were immunostained using rabbit anti-alpha smooth muscle actin (ab5694 1:200; Abcam, Cambridge, MA, USA) primary antibody for 24 h at 4 °C. Sections were washed for 10 min in TBS (three times), incubated in Alexa Fluor 488 go...
To predict gene targets associated with vascular fragility and CMHs, we used the IRIDESCENT (Wren & Garner, ) text mining package, as described (Toth et al.,, ). Using this method, important genes relevant for the pathogenesis of CMHs and structural integrity of the vasculature were identified (Table ). The mRNA expression of these genes in cerebral arteries isolated on day 10 post-induction of hypertension was analyzed by a quantitative real-time RT-PCR technique using a Strategen MX3000 platform, as previously reported (Toth et al., ). In brief, total RNA was isolated with a Mini RNA Isolation Kit (Zymo Research, Orange, CA, USA) and was reverse-transcribed using Superscript III RT (Invitrogen). Amplification efficiencies were determined using a dilution series of a standard vascular sample. Quantification was performed using the efficiency-corrected ΔΔCq method. The relative quantities of the reference genes Hprt, Ywhaz, B2m, Gapdh, Actb, and S18 were determined, and a normalization factor was calculated based on the geometric mean for internal normalization. Fidelity of the PCR was determined by melting temperat...
Machine-readable layer
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