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Nov 2017
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Induction of Photothrombotic Stroke in the Sensorimotor Cortex of Rats and Preparation of Tissue for Analysis of Stroke Volume and Topographical Cortical Localization of Ischemic Infarct
大鼠感觉运动皮层脑卒中的诱导性光栓疗法及用于卒中体积分析和缺血性梗死地形图皮层定位的组织制备   

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Abstract

The photothrombotic model of stroke is commonly used in research as it allows the ischemic infarct to be targeted to specific regions of the cortex with high reproducibility and well-defined infarct borders. Unlike other models of stroke, photothrombosis allows the precise size and location of infarct to be tightly controlled with minimal surgical invasion. Photothrombosis is induced when a circulating photosensitive dye is irradiated in vivo, resulting in focal disruption of the endothelium, activation of platelets and occlusion of the microvasculature (Watson et al., 1985; Dietrich et al., 1987; Carmichael, 2005). The protocols here define how photothrombosis can be specifically targeted to the sensorimotor forelimb cortex of rat with high reproducibility. Detailed methods on rat cortical tissue processing to allow for accurate analysis of stroke volume and stereotactic determination of the precise cortical region of ischemic damage are provided.

Keywords: Photothrombosis (光栓疗法), Ischemic infarct (缺血性梗死), Stroke model (卒中模型), Stroke volume (卒中体积), Ischemia (缺血)

Background

The photothrombotic model of stroke allows for the precise placement of an ischemic infarct in specific regions of the cortex (Carmichael, 2005; Underly and Shih, 2017). Photothrombosis can be used to occlude specific arteries and arterial branches in the cortex (Carmichael et al., 2005), individual vessels of the pia (Taylor and Shih, 2013) and defined cortical areas such as the barrel field (Dietrich et al., 1987) and hind limb somatosensory cortex (Que et al., 1999). Using this approach, highly reproducible ischemic infarcts have been generated in many experimental animal models including rodents (Watson et al., 1985; Carmichael et al., 2005) and non-human primates (Ikeda et al., 2013). Photothrombosis of the forelimb sensorimotor cortex is useful as it results in localized sensorimotor impairment in forelimb use that can be carefully quantified using a variety of behavioral tests after stroke and during recovery (Sist et al., 2014; Wiersma et al., 2017). The methods presented here allows for the induction of consistent ischemic infarcts of the forelimb sensorimotor cortex that result in significant and long-lasting deficits in forelimb motor function (Wiersma et al., 2017). There is currently no standardized method of identifying both the volume of induced stroke and the anatomical location of stroke in the cortex. Here, we provide methods on systematic identification of the precise cortical location of photothrombotic infarct and analysis of stroke volume. Exclusion criteria for animal stroke models are widely varied. Therefore we provide guidelines to establish criteria for exclusion of animals that deviate from expected stroke volumes or stereotaxic locations.

Materials and Reagents

  1. Surgical
    1. Sterile scalpel blade # 10 (Fine Scientific Tools, catalog number: 10100-00 )
    2. Blunt 16-gauge needle (STEMCELL Technologies, catalog number: 28110 )
    3. 1 ml syringes x 5 (BD, catalog number: 309659 )
    4. I.V. catheter 24G x 5/8” (Smiths Medical, Jelco®, catalog number: 4073 )
    5. Silk suture 5-0, P-3 reverse cutting (Ethicon, PERMAHAND®, catalog number: 640G )
    6. Sprague-Dawley rat, male, ~500 g, 15-20 weeks of age (Charles River)
    7. Isoflurane USP 99% (Fresenius Kabi, catalog number: CP0406V2 )
    8. Compressed oxygen gas with high purity 99.995% (Praxair)
    9. Compressed nitrous oxide gas 99% purity (Praxair)
    10. BetadineTM surgical scrub (Fisher Scientific, catalog number: 19-027132)
      Manufacturer: Purdue Pharma, catalog number: 6761815117 .
    11. Sterile saline, 0.9% NaCl (Baxter, catalog number: 2B1324X )
    12. Rose bengal (Sigma-Aldrich, catalog number: 330000 )
    13. 0.25% Bupivacaine (Sigma-Aldrich, catalog number: B5274 )
    14. Buprenorphine (Schering- Plough, 0.2 mg injectable)
    15. Rose bengal solution (see Recipes)

  2. Perfusion
    1. Euthasol® (VIRBAC, catalog number: 710101 )
    2. Celline isotonic saline solution (Fisher Scientific, catalog number: 351142-10)
      Manufacturer: SCP SCIENTIFIC, catalog number: CS20310D .
    3. Heparin (Sigma-Aldrich, catalog number: H3393 )
    4. Formalin 1:10 dilution, buffered (Fisher Scientific, catalog number: SF100-20 )
    5. Isotonic saline heparin (5,000 IU/L) solution (see Recipes)
    6. 4% formalin solution (see Recipes)

  3. Tissue Cryosectioning
    1. Superfrost® plus gold slides (Fisher Scientific, catalog number: 22-035813 )
    2. 2-methylbutane (Sigma-Aldrich, catalog number: 277258 )
    3. D-Sucrose (Fisher Scientific, catalog number: 10638403 )
    4. Tissue Tek® OCT compound (Sakura, catalog number: 4583 )
    5. 30% sucrose solution (see Recipes)

  4. Stroke Analysis
    1. Rat atlas (Paxinos, George, and Charles Watson. The rat brain in stereotaxic coordinates: hard cover edition. Access Online via Elsevier, 2006. A tool by Matt Gaidica.)

Equipment

  1. Heating pad with rectal thermometer (Stoelting, catalog number: 50300 )
  2. Sterile Crile hemostat (Fine Science Tools, catalog number: 13004-14 )
  3. Sterile blunt ended scissors (Fine Science Tools, catalog number: 14001-18 )
  4. Sterile rongeur (Fine Science Tools, catalog number: 16000-14 )
  5. Carbide burs drill bit (SS WHITE, catalog number: 14002-5 )
  6. Perfusion tray (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 11469 )
  7. Sterile scoop (Fine Science Tools, catalog number: 10090-13 )
  8. Weigh scale (Kent Scientific, catalog number: SCL-1015 )
  9. Electric razor, Mini cordless trimmer (Braintree Scientific, catalog number: CLP-9868 )
  10. Animal anesthetic system (Harvard Apparatus, catalog number: 72-6467 )
  11. Anesthetic induction chamber (Smiths Medical, catalog number: V711801 )
  12. Stereotaxic frame (KOPF INSTRUMENTS, model: Model 963 Standard Accessories)
  13. Surgical drill–TCM Endo III (Nouvag, catalog number: 31817 )
  14. Laser 532 nm (Laserglow Technologies, model: LCS-0532 , Power Supply, Laserglow Technologies, model: C5310051X , with Thorlabs dielectric mirror to focus laser, Thorlabs, catalog number: CM1-E02 )
  15. Laser safety goggles (Laserglow Technologies, catalog number: AGF5565XX )
  16. Digital Peri-Star Peristaltic Pump (World Precision Instruments, catalog number: PERIPRO-4HS )
  17. Cryostat (Leica, model: CM3050 S Research Cryostat)
  18. Leica HCX PL APO L 10x 1.0NA water emersion objective lens
  19. Leica SP5 Confocal microscope (Leica, model: Leica SP5 )
  20. Animal surgery stereo microscope (Leica, M-series)
  21. Inverted microscope (Leica, model: Leica DMI6000B )

Software

  1. Leica LAS AF Software
  2. ImageJ
  3. Prism by Graph Pad vs9

Procedure

  1. Solution preparation
    1. Prior to beginning the surgical preparation make rose bengal solution (Recipe 1) to 100 mg/ml in sterile saline.
      Note: Ensure that the solution is not exposed to light and that the powder is not exposed to light during storage or while making the solution. Rose bengal solution should be made fresh prior to injection.

  2. Anesthetic and surgical preparation
    1. Weigh animal and record weight.
    2. Place a rat in an anesthetic induction chamber.
    3. Adjust the oxygen flow rate to 1.5 L/min and adjust isoflurane vaporizer to 3%, gradually increase isoflurane to 5% over 2 min.
    4. Once the animal losses righting reflex, stop gas flow to the chamber.
    5. Rapidly remove the rat from the induction chamber and place in anesthetic mask or nosecone.
    6. Immediately redirect flow to an anesthetic nosecone, reduce oxygen flow rate to 0.3-0.4 L/min, then set nitrogen flowmeter to 0.6-.08 L/min and reduce the isoflurane to 1.5 to 2.0%.
      Note: We use 1.5-2.0% isoflurane (in 70% nitrous oxide and 30% oxygen) at a flow rate of 1 L/min to maintain anesthetic plane in 500 g rats. Anesthetic and inhaled gas choices are important to consider when inducing stroke as oxygenation and vasoconstriction can alter the size of induced stroke.
    7. Ensure the animal is in surgical plane of anesthetic as evident by a loss of reflexes, muscles relaxation and deep rhythmic breathing in accordance with IACUC protocols.
    8. Ensure body temperature is maintained at 37 °C with a rectal temperature probe and a heating pad.
    9. Apply ophthalmic ointment to both eyes to prevent damage to the cornea, and reapply every 15 min or as needed.
    10. Remove hair from the surgical field on the rat scalp from the ears to the eyes by cutting hair at the root right next to the skin with a pair of sterile surgical scissors or fur can be shaven with an electric razor.
    11. Prepare skin with Betadine® surgical scrub.

  3. Thin window of the skull
    1. Mount the animal onto the stereotaxic device (Figure 1A).
    2. Start by placing one ear bar in the ear canal and tighten into place, then slide the other ear bar into the ear canal until the head can no longer move from left to right. Tighten the second ear bar into place (Figures 1B and 1C).
      Note: The ear bars have been placed correctly if the head does not shift left to right.
    3. Secure the anterior mount or bite bar in the mouth of the rat. Adjust the bite bar until the head is level. The rat is properly secured in the stereotaxic apparatus then the head does not move in any direction when pressure is applied (Figure 1D).
      Note: Proper placement of the rat in the stereotaxic frame is essential as drilling and thinning of the skull require that there is no movement of the head.


      Figure 1. Mounting of rats in a stereotaxic surgical device. A. Standard stereotaxic head frame for rodents, with adjustable ear bars and anesthetic nosecone over the bite bar. B. Rat mounted in a stereotaxic frame with secured ear bars. C. Proper placement of rat head in stereotaxic frame such that the head is centered in the frame as indicated by red dashed lines. D. Proper adjustment of nosecone and bite bar height to ensure the skull is level as indicated by yellow dashed lines.

    4. Focus the surgical area under an animal surgery stereo microscope at 5x.
    5. Induce an incision line block with 0.25% Bupivacaine (do not exceed 8 mg/kg).
    6. Make a 1 cm incision over the skull midline (Figure 2A), superior to the ear and inferior to the eyes.
    7. Expose the skull by retracting each side of the incision away from the midline.
    8. Bregma can now be easily located (Figure 2B), if you have difficulty locating bregma gently push on the skull plate and the sutures will become obvious as they shift under mild pressure.
    9. Mark the stereotaxic coordinates which overly the preferred forelimb sensory motor cortex (Figure 2C) at the following points relative to bregma (0,0): (1,3), (1,-1), (4,3) and (4,-1)
    10. Thin the skull in a rectangle between the marked coordinates (Figures 2D and 2E), 1-4 mm lateral; -1 to +3 mm anterior of bregma, using a surgical drill with endodontic electronic motor system set to a speed of 5,000 rpm fitted with a dental handset fitted and rounded carbide bur drill bit designed for cutting. The skull should be thinned to a thickness of 0.1 mm uniformly over the 3 x 4 mm area.
      Note: The vasculature underlying the thinned skull should be clearly visible. If you cannot clearly see the underlying vasculature, try adding a drop of sterile saline over the window to clear the window of any particulate generated by the drilling process. If the underlying vascular is not visible after adding a drop of saline, additional thinning is needed, continue thinning the skull until the vascular becomes visible. 


      Figure 2. Photothrombosis of the sensorimotor cortex in rats. A. Location of midline incision to exposed the skull (indicated by the red dashed line) of a rat mounted in stereotaxic frame. B. Surgically exposed skull with; C. the stereotactic coordinates of the thin window corners marked by black circles, established relative to bregma (marked by the yellow circle) using a surgical microscope (5x magnification). D. Thin window of the skull (indicated by the black box) created between the coordinates outlined in; C. Blood vessels under the thinned skull become visible at 5x magnification; E. F. Laser targeted (indicated by green circle) to the blood vessels directly under the thin window to induce an ischemic infarct of the sensorimotor cortex, shown at 5x magnification in; G.

  4. Tail vein injection of photoactive dye
    1. Calculate the volume of rose bengal (prepared prior to surgery Step A1) needed for injection at a concentration of 30 mg/kg as follows:



    2. Swab rat tail with warm water to increase visibility of the vein (Figures 3A and 3B).
    3. Occlude the vein with non-dominant hand (Figure 3C).
    4. Slide the needle into the tail vein with the bevel facing upward and the needle directed parallel to the vein (Figures 3D and 3E).
    5. A flash of blood indicates the catheter is in the correct location (Figure 3F).
    6. Slide the catheter into the vein and remove the needle (Figure 3G).
    7. Ensure there is blood flowing out of the catheter (Figure 3F).
    8. Attach needle with rose Bengal.
    9. Inject calculated volume (D1) of rose bengal at a rate of 1 ml/min.
    10. Flush the catheter with 0.2 ml of sterile saline.
      Note: Rose bengal can move into the perivascular space following injection leading to ischemia in the tail vein and subsequent necrotic cell death, a saline flush can prevent this complication.


      Figure 3. Tail vein injection in rats. A. Location of rat tail veins marked with red dashed lines. B. Rotate rat tail 90° so that the tail vein becomes visible. C. Occlude the vein with non-dominant hand. D. With the bevel facing upward align the catheter needle with tail vein. E. Direct the needle parallel to the vein. F. Insert the needle until a flash of blood is visible in the catheter. G. Remove the needle leaving the catheter in the vein. H. Check to ensure blood flow out of the catheter to ensure correct placement. The catheter is now ready for injection. I. After injection remove catheter. J. Apply pressure to allow a clot to form. K. Ensure that the tail looks healthy and blood flow has returned to the tail tip.

  5. Induction of photothrombosis
    1. Put on laser safety glasses.
    2. Immediately illuminate the thinned skull over the somatosensory cortex using a collimated beam of green laser light (532 nm, 17 mW; ~4.0 mm beam diameter) for 15 min to photoactivate the Rose Bengal (Figures 2F and 2G), occluding all illuminated cortical vasculature and inducing a focal ischemic infarct.
      Note: The precise size and location of the laser beam placement will determine the size and location of photothrombotic stroke. The laser beam is turned on immediately after the tail vein injection flush (Step D3), for sham surgical animals follow all of the outlined steps but simply do not turn on the laser during the 15 min exposure time period (skip Step E2).
    3. Following the 15 min of laser exposer, suture skull and give analgesic care in accordance with IACUC protocols (0.05 mg/kg subcutaneous buprenorphine).
    4. Remove tail vein catheter.
    5. Give a subcutaneous injection of 3 ml sterile saline to prevent dehydration.
    6. After surgery when mobility is regained, observe animal for signs of paralysis and weakness in the forelimb associated with the injured cortex.
      Note: There should be a clear deficit in the injured limb immediately after the procedure. In most cases, this is evident by the rat avoiding use of the stroke lesioned forepaw by lifting and clenching the injured paw while using only the uninjured forepaw for postural support of body weight.

  6. Transcardial perfusion of rat
    1. Prepare perfusion solutions (4% formalin and Saline with heparin) and allow time to reach correct temperature for use (Recipe 2).
      Note: See Gage et al. (2012) for a detailed protocol on rodent perfusion.
    2. Intraperitoneally inject sodium pentobarbital at 150 mg/kg in accordance with the American Veterinary Medical Association Guidelines for Euthanasia.
      Note: For a 500 g rat the volume of Euthasol® (400 mg/ml sodium pentobarbital) to be injected would be calculated as follows:



      Example for 500 g rat: Convert rat weight to kg: 500 g = 0.50 kg. Determine the mass in mg of sodium pentobarbital to inject: 0.500 kg x 150 mg/kg = 75 mg. Determine the volume of Euthasol® to inject it is sold at (400 mg/ml): 75 mg/400 mg/ml = 0.188 ml.
    3. Wait for surgical plane to be reached then secure animal on its back to a perfusion tray set at 85° (Figure 4A).
    4. Locate the xiphoid process following the xiphoid plane make a transverse incision through the integument and abdominal wall following just beneath the rib cage.
    5. Make an incision through the diaphragm using blunt-ended scissors and continue the incision along the entire rib cage exposing the pleural cavity.
    6. Make sagittal incisions through both sides of the rib cage, ensuring not to damage the heart or lungs.
    7. Lift and pin the rib cage to reveal the coronal chest and release the heart of any attachments to the chest cavity (Figure 4B).
    8. Using a hemostat, grip the apex of the heart and insert a blunt ended 16-gauge needle through the left apex, continue insertion into the left ventricle (Figure 4C).
      Note: It is important to ensure that the needle does not extend past the aortic arch.
    9. Make a small incision in the right atria to release fluid pressure and start primed peristaltic perfusion pump at 70-100 ml/min (Figure 4D).
    10. Perfuse 250 ml of 37 °C isotonic saline (Celline) Heparin (5,000 IU/L) solution (Recipe 3) to clear all blood from the animal.
      Note: The animal should become pale, the liver should be white and only clear fluids should be draining from the right atrial in successful perfusions.
    11. Switch perfusion fluid to 4% formalin solution at 4 °C, again perfuse 250 ml of the solution at a rate of 70-100 ml/min.
      Note: Fixative tremors should begin to occur throughout the rat within seconds of the 4% formalin solution being infused and the rat should be very stiff by the end of the perfusion.


      Figure 4. Transcranial perfusion of rat. A. Rat placed on perfusion tray set at 85°, dashed lines indicate the location of incisions. B. Surgically exposed coronal chest with heart highlighted. C. Insertion of blunt ended 16-gauge needle through the left apex of the heart. D. Incision into the right atria to release fluid pressure and drain perfusion fluids.

  7. Collection of rat brain
    1. Decapitate the animal between the cervical spinal cord (C1) and the skull.
    2. Create a midline incision spanning the skull and remove all skin to reveal the whole skull.
    3. Insert a rongeur into the void where the cervical vertebra has been detached sliding it along the occipital skull.
    4. Cut through the occipital skull and pull the bone away from the hindbrain, repeat on the other side.
    5. Insert a blunt scissor under the skull along the midline, cutting the skull at the midline.
    6. Peel each half of the skull away from the brain.
    7. Clip any sharp edges remaining as the brain is now ready to be removed from the open skull cavity.
    8. Insert a small scoop between the frontal lobe and olfactory bulbs releasing the brain from the bulbs.
    9. Run the scoop under the brain until all connecting tissue blood vessels are cleared and the brain easily slides out of the open skull.

  8. Tissue fixing, freezing and cryosectioning
    1. Immediately following removal, place the brain in 50 ml of 4% formalin, leave overnight at 4 °C.
    2. After overnight fixation, replace 4% formalin with 4 °C 50 ml of 30% sucrose solution (Recipe 4) and leave tissue for 3-4 days at 4 °C until the brain is no longer buoyant in solution.
    3. Remove tissue from solution (tissue will be stiff and white in color).
    4. Cover the brain with a thin layer of OCT freezing media.
    5. Submerge the brain in 2-methylbutane solution chilled to -50 °C.
    6. Leave the brain submerged for 1 min to ensure the brain is frozen throughout.
      Note: If the whole brain will be stored for an extended time (more than 2 weeks), the brain should be coated in an additional layer of OCT freezing media.
    7. The tissue is now ready for long-term storage at -20 °C or immediate cryosectioning.
      Note: If planning to section the tissue, we do not recommend storage at -80 °C, as the tissue must be warmed to -20 °C for sectioning. We experienced fracturing of tissues and the appearance of freeze-thaw line when we warmed out tissue to optimal sectioning temperature after storage at -80 °C.
    8. A cryostat should be set to a temperature between -19 °C and -22 °C, with a cutting thickness of 20 μm for coronal brain slices.
    9. Section the whole brain and mount all slices on glass microscope slides.
      Note: If Immunochemistry will be performed Superfrost® Plus Gold slides are recommended as we found slices did not adhere well to uncoated slides even after baking.
    10. Slides can now be counterstained or immunolabeled.

  9. Microscopy of stroke lesion
    1. Select 10x microscope objective lens on confocal microscope.
      Note: We use a Leica SP5 Confocal microscope with Leica HCX PL APO L 10x 1.0NA water emersion objective lens and Leica LAS AF Software.
    2. The Stroke region can be visualized with a setting for an applied counterstain, immunolabel or in some cases autofluorescence in the FITC emission spectrum can be used to effectively define the stroke borders. In unstained tissue, the stroke border can be determined using the light differential interference contrast or light-DIC setting.
    3. Using microscope software set up parameters to collect tile scan images encompassing the whole brain slice (Figure 5A).
    4. Collect images of each brain slice spanning the infarct (Figure 5B).


      Figure 5. Coronal sections spanning the ischemic infarct. A. Coronal sections of rat brain with the lateral coordinates relative to bregma, the infarct area is shown in red. B. The stroke area corresponding to each coronal slice shown in (A) visualized using spectral settings for the FITC emission spectrum on the confocal microscope at 10x magnification. Adapted from Wiersma et al., 2017.

  10. Volume analysis of ischemic lesion
    1. Cortical stoke volume can be determined by analyzing the volume of infarct in each coronal section spanning the ischemic lesion (Figure 5).
    2. Import collected images (Procedure I) from each coronal section spanning the stroke imported into ImageJ (Figure 6A).
    3. Using the measure function in ImageJ, trace the infarct on each section to determine the specific surface area of the stroke in each slice (Figure 6B).
    4. Multiply the surface area by the depth of slice (20 μm) to determine the lesion volume for each slice (Figure 6C).


      Figure 6. Example calculation of infarct volume for one coronal section. A. The infarct is clearly visible in the slice visualized using spectral settings for the FITC emission spectrum on the confocal microscope at 10x magnification. B. The area of the infarct to trace in ImageJ is shown by the white dashed line. C. ImageJ calculates the surface area of the infarct as outlined in (B), which is multiplied by the slice thickness to provide the stroke volume in the slice.

    5. The infarct volumes for every slice spanning the lesion can be summed to determine the total stroke volume.
    6. It is important to predetermine exclusion criteria and remove any animals from the study which do not have a minimum predetermined stroke volume (see Data analysis).

  11. Topographical cortical mapping of ischemic infarct
    1. Cortical maps showing the topographical location of induced stroke are created for each animal by recording the stereotaxic coordinates of stroke boundaries for each coronal slice of tissue spanning the lesion.
      For each coronal slice spanning the lesion, use a rat atlas to determine the y coordinate of the slice (Figure 7A).
    2. Determine the two x coordinates of the stroke boundaries at the established y coordinate (Figure 7A).
    3. Plot the coordinates on a grid (Figure 7B).
    4. Once all the points are plotted, they can be joined to create an outline of the stroke area (Figure 7C).


      Figure 7. Finding coordinates for topographic infarct map of the stroke cortex. A. A coronal slice is overlaid on the rat atlas to determine the y coordinates of the stroke, then the x coordinates of the stroke boundary are determined for the slice (red circles). B. The boundary coordinates are transferred onto on to a grid (red circles). This is repeated until all the coordinates are entered and a line can be drawn between points to outline the stroke boundary. C. The grid can then be overlaid on the cortex to demonstrate the stroke location. Adapted from Wiersma et al., 2017.

    5. An average topographical map of the stroke area can be created by averaging the stereotactic x coordinates for each y coordinate of the stroke boundaries or the stroke areas can be overlaid to show constancy of infarct size and stereotaxic location.
    6. The acceptable location of stroke should be predetermined and animals which have strokes outside of the accepted margins should be removed from the study (see Data analysis).

Data analysis

  1. Photothrombotic stroke is highly consistent in volume and location of infarct. We performed n = 52 strokes in male Sprague Dawley Rats and found an average stroke size of 4.424 mm3 with a standard deviation of 0.316 and standard error of 0.0432 (Figure 8A). We would recommend excluding animals with a stroke volume outside of 3 standard deviations from the mean stroke volume.
  2. When the topographical representations of all 52 strokes are overlaid, it becomes clear that the location of stroke in the cortex was very constant (Figure 8B). The average stroke topographical map can be created by averaging the x and y coordinates at each point. We would recommend excluding any animal which has coordinate 2 mm away from the average coordinate at any location.
  3. When comparing the stroke size between different groups, we used a student’s unpaired two-tailed t-testing to determine if the stroke volumes between groups were significantly different.


    Figure 8. Data analysis of stroke volume and location. A. Stroke volume of n = 52 animals, with the mean indicated by the long central bar and the standard error bar shown on either side of the mean. B. Topographical cortical map of n = 52 strokes overlaid. Adapted from Wiersma et al., 2017.

Notes

    The photothrombotic stroke model is very reproducible, such that none of the 52 animals given strokes in our study had to be excluded for having strokes volumes more than 3 standard deviations away from the mean. All of the animals receiving strokes in our study showed significant behavioral deficits as a result of the ischemic lesion.

Recipes

  1. Rose bengal solution
    1. Weigh out 100 mg of rose bengal in a small tube
    2. Measure 1 ml sterile saline
      Note: All weighing and measuring steps should be done in minimal light.
    3. Add saline to rose bengal, mix vigorously until all rose bengal is in solution.
      Note: Once rose bengal is in solution it is very light sensitive, and any light exposure should be avoided. Rose Bengal solution should be used within 4 h after making it.
  2. 4% formalin solution
    1. Measure 300 ml isotonic saline
    2. Add 200 ml of 1:10 formalin solution
    3. Invert to mix
    4. Chill to 4 °C
  3. Isotonic saline heparin (5,000 IU/L) solution
    1. Measure 500 ml isotonic saline
    2. Warm saline to 37 °C
    3. Add 2,500 IU heparin
      Note: Heparin will denature overtime at 37 °C, do not add until right before use.
    4. Invert to mix heparin into saline
  4. 30% sucrose solution
    1. Measure 45 ml of isotonic saline
    2. Add 15 g of D-sucrose
    3. Mix vigorously
    4. Chill to 4 °C

Acknowledgments

This work was supported by Alberta Innovates Health Solutions, Heart and Stroke Foundation of Canada, Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada. Grants from the Canada Foundation for Innovation and Province of Alberta Small Equipment Grants were used to acquire confocal microscopy equipment. This protocol and figures are adapted from previous work: Wiersma, A. M., Fouad, K. and Winship, I. R. (2017). Enhancing spinal plasticity amplifies the benefits of rehabilitative training and improves recovery from stroke. J Neurosci 37(45): 10983-10997. The authors have no conflicts of interest to declare.

References

  1. American Veterinary Medical Association Guidelines for Euthanasia (2013) (AVMA).
  2. Carmichael, S. T. (2005). Rodent models of focal stroke: size, mechanism, and purpose. NeuroRx 2(3): 396-409.
  3. Carmichael, S. T., Archibeque, I., Luke, L., Nolan, T., Momiy, J. and Li, S. (2005). Growth-associated gene expression after stroke: evidence for a growth-promoting region in peri-infarct cortex. Exp Neurol 193(2): 291-311.
  4. Dietrich, W. D., Watson, B. D., Busto, R., Ginsberg, M. D. (1987). Metabolic plasticity following cortical infarction: a 2-deoxyglucose study. In: Raichel, M. E. and Powers, W. J. (Eds). Cerebrovascular disorders. Raven Press pp: 285-295.
  5. Gage, G. J., Kipke, D. R. and Shain, W. (2012). Whole animal perfusion fixation for rodents. J Vis Exp (65).
  6. Ikeda, S., Harada, K., Ohwatashi, A., Kamikawa, Y., Yoshida, A. and Kawahira, K. (2013). A new non-human primate model of photochemically induced cerebral infarction. PLoS One 8(3): e60037.
  7. Que, M., Schiene, K., Witte, O. W. and Zilles, K. (1999). Widespread up-regulation of N-methyl-D-aspartate receptors after focal photothrombotic lesion in rat brain. Neurosci Lett 273(2): 77-80.
  8. Sist, B., Fouad, K. and Winship, I. R. (2014). Plasticity beyond peri-infarct cortex: spinal up regulation of structural plasticity, neurotrophins, and inflammatory cytokines during recovery from cortical stroke. Exp Neurol 252: 47-56.
  9. Taylor, Z. J. and Shih, A. Y. (2013). Targeted occlusion of individual pial vessels of mouse cortex. Bio-Protocol e897.
  10. Underly, R. G. and Shih, A. Y. (2017). Photothrombotic induction of capillary ischemia in the mouse cortex during in vivo two-photon imaging. Bio-protocol e2378.
  11. Watson, B. D., Dietrich, W. D., Busto, R., Wachtel, M. S. and Ginsberg, M. D. (1985). Induction of reproducible brain infarction by photochemically initiated thrombosis. Ann Neurol 17(5): 497-504.
  12. Wiersma, A. M., Fouad, K. and Winship, I. R. (2017). Enhancing spinal plasticity amplifies the benefits of rehabilitative training and improves recovery from stroke. J Neurosci 37(45): 10983-10997.

简介

中风的光血栓形成模型通常用于研究,因为它允许缺血性梗塞针对特定区域的皮层,具有高重现性和明确的梗塞边界。 与其他卒中模型不同,光血栓形成能够以最小的手术入侵严密控制梗死的精确大小和位置。 当在体内辐射循环的光敏染料时诱导光血栓形成,导致内皮的局灶性破坏,血小板的活化和微血管的闭塞(Watson等人,1985年 ; Dietrich等人,1987; Carmichael,2005)。 这里的方案定义了如何将光致血栓形成特异性地靶向大鼠的感觉运动前肢皮层并具有高重现性。 提供了大鼠皮质组织处理的详细方法以允许精确分析脑卒中体积和立体定向确定缺血性损伤的精确皮层区域。

【背景】中风的光血栓形成模型允许在皮层的特定区域中精确布置缺血性梗塞(Carmichael,2005; Underly and Shih,2017)。光血栓形成可用于封闭皮层中的特定动脉和动脉分支(Carmichael et al。,2005),pia的个别血管(Taylor和Shih,2013),并定义了皮质区域如桶(Dietrich等人,1987)和后肢躯体感觉皮层(Que等人,1999)。使用这种方法,在许多实验动物模型中已经产生了高度可重现的缺血性梗塞,包括啮齿动物(Watson等人,1985; Carmichael等人,2005)和非人类动物模型人灵长类动物(Ikeda et al。,2013)。前肢感觉运动皮层的光血栓形成是有用的,因为其导致前肢使用中的局部感觉运动损伤,其可以使用中风后和恢复期间的各种行为测试来仔细量化(Sist et al。,2014; Wiersma et al。,2017)。本文提出的方法允许诱发前肢感觉运动皮层的一致性缺血性梗塞,导致前肢运动功能显着且持久的缺陷(Wiersma等人,2017)。目前还没有标准的方法来确定诱发中风的体积和皮质中中风的解剖位置。在这里,我们提供系统确定光凝血梗塞的精确皮层位置和分析脑卒中体积的方法。动物卒中模型的排除标准各不相同。因此,我们提供指导方针,以建立排除偏离预期中风量或立体定位位置的动物的标准。

关键字:光栓疗法, 缺血性梗死, 卒中模型, 卒中体积, 缺血

材料和试剂

  1. 外科
    1. 无菌手术刀刀片#10(Fine Scientific Tools,目录号:10100-00)
    2. 钝的16号针头(STEMCELL Technologies,目录号:28110)
    3. 1毫升注射器×5(BD,目录号:309659)
    4. 静脉注射导管24G×5/8“(Smiths Medical,Jelco ,目录号:4073)
    5. 丝线5-0,P-3反向切割(Ethicon,PERMAHAND ®,目录号:640G)
    6. Sprague-Dawley大鼠,雄性,〜500克,15-20周龄(Charles River)
    7. 异氟醚USP 99%(Fresenius Kabi,目录号:CP0406V2)
    8. 高纯度压缩氧气99.995%(普莱克斯)
    9. 压缩的一氧化二氮气体纯度达99%(普莱克斯)
    10. Betadine TM手术磨砂(Fisher Scientific,目录号:19-027132)
      制造商:Purdue Pharma,目录号:6761815117 。
    11. 无菌盐水,0.9%NaCl(Baxter,目录号:2B1324X)
    12. 玫瑰孟加拉(Sigma-Aldrich,目录号:330000)
    13. 0.25%布比卡因(Sigma-Aldrich,目录号:B5274)
    14. 丁丙诺啡(先灵葆雅,0.2毫克注射剂)
    15. 玫瑰孟加拉解决方案(见食谱)

  2. 灌注
    1. Euthasol (VIRBAC,目录号:710101)
    2. 咖啡因等渗盐溶液(Fisher Scientific,目录号:351142-10)
      制造商:SCP SCIENTIFIC,目录号:CS20310D
    3. 肝素(Sigma-Aldrich,目录号:H3393)
    4. 福尔马林1:10稀释,缓冲(Fisher Scientific,目录号:SF100-20)
    5. 等渗盐水肝素(5,000 IU / L)溶液(见食谱)
    6. 4%福尔马林溶液(见食谱)

  3. 组织冷冻切片
    1. Superfrost 加上金色幻灯片(Fisher Scientific,目录号:22-035813)
    2. 2-甲基丁烷(Sigma-Aldrich,目录号:277258)
    3. D-蔗糖(Fisher Scientific,目录号:10638403)
    4. 组织Tek OCT化合物(Sakura,目录号:4583)
    5. 30%蔗糖溶液(见食谱)

  4. 中风分析
    1. 大鼠地图集(Paxinos,George和Charles Watson,立体定位坐标中的大鼠脑:硬封面版,通过Elsevier访问在线,2006年,由Matt Gaidica提供的工具)

设备

  1. 直肠温度计加热垫(Stoelting,目录号:50300)
  2. 无菌Crile hemostat(Fine Science Tools,目录号:13004-14)
  3. 无菌钝头剪刀(Fine Science Tools,目录号:14001-18)
  4. 无菌咬骨钳(精细科学工具,目录号:16000-14)

  5. 硬质合金钻头钻头(SS WHITE,目录号:14002-5)
  6. 灌注盘(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:11469)
  7. 无菌瓢(精细科学工具,目录号:10090-13)
  8. 称重秤(Kent Scientific,目录号:SCL-1015)
  9. 电动剃须刀,迷你无线修剪器(Braintree Scientific,目录号:CLP-9868)
  10. 动物麻醉系统(哈佛仪器,目录号:72-6467)
  11. 麻醉诱导室(Smiths Medical,目录号:V711801)
  12. 立体定向框架(KOPF INSTRUMENTS,型号:963型标准配件)

  13. 手术钻 - TCM Endo III(Nouvag,产品目录号:31817)
  14. 激光532 nm(Laserglow Technologies,型号:LCS-0532,电源,Laserglow Technologies,型号:C5310051X,带Thorlabs介质镜聚焦激光器,Thorlabs,产品目录号:CM1-E02)
  15. 激光护目镜(Laserglow Technologies,目录号:AGF5565XX)
  16. 数字周星蠕动泵(世界精密仪器公司,产品目录号:PERIPRO-4HS)
  17. 低温恒温器(Leica,型号:CM3050 S Research Cryostat)
  18. 徕卡HCX PL APO L 10x 1.0NA水再现物镜
  19. Leica SP5共聚焦显微镜(Leica,型号:Leica SP5)
  20. 动物手术体视显微镜(徕卡,M系列)
  21. 倒置显微镜(徕卡,型号:徕卡DMI6000B)

软件

  1. 徕卡LAS AF软件
  2. ImageJ
  3. 通过Graph Pad vs9的棱镜

程序

  1. 溶液准备
    1. 在开始手术前,将孟加拉玫瑰红溶液(配方1)置于无菌生理盐水中100 mg / ml。
      注意:确保溶液不会暴露在光线下,并且在储存期间或制作溶液时粉末不会暴露在光线下。注射前应将玫瑰孟加拉溶液制成新鲜。

  2. 麻醉和手术准备
    1. 称动物并记录体重。
    2. 将一只老鼠放入麻醉诱导室。
    3. 调整氧气流速至1.5 L / min,并将异氟醚蒸发器调整至3%,在2分钟内将异氟醚逐渐增加至5%。

    4. 一旦动物失去正确的反射,停止气体流入室。

    5. 快速将大鼠从诱导室中取出并放入麻醉面罩或鼻锥中。
    6. 立即将流量重定向至麻醉剂鼻锥,将氧气流速降至0.3-0.4 L / min,然后将氮气流量计设置为0.6-0.08 L / min,并将异氟醚降低至1.5-2.0%。
      注:我们使用1.5-2.0%异氟醚(70%氧化亚氮和30%氧气),流量为1 L / min,以保持500 g大鼠的麻醉平面。
      吸氧和血管收缩可以改变诱发中风的大小,因此麻醉和吸入气体的选择在诱发中风时很重要。
    7. 根据IACUC协议,确保动物处于麻醉的手术平面,如反射消失,肌肉松弛和深度有节奏的呼吸。

    8. 使用直肠温度探头和加热垫确保体温保持在37°C。

    9. 在两只眼睛上涂抹眼药膏以防止损伤角膜,并且每15分钟或根据需要重新涂抹。

    10. 用一把无菌外科剪刀在皮肤旁边的根部切割头发,将头发从大鼠头皮上的手术区域从耳朵移至眼睛,或者用电动剃须刀刮毛。
    11. 用Betadine ®手术磨砂准备皮肤。

  3. 头骨的薄窗
    1. 将动物装到立体定位装置上(图1A)。
    2. 首先将一个护耳放入耳道并拧紧,然后将另一个护耳滑入耳道,直至头部不能再从左向右移动。将第二个耳杆拧紧到位(图1B和1C)。
      注意:如果头部不从左向右移动,则耳条已正确放置。
    3. 将前牙或咬合棒固定在老鼠的嘴中。调整咬合杆直到头部水平。大鼠被适当地固定在立体定位仪器中,然后在施加压力时头部不会朝任何方向移动(图1D)。
      注意:正确放置大鼠在立体定位框架中是至关重要的,因为钻孔和头骨变薄需要头部没有移动。


      图1.鼠标在立体定位手术设备中的安装A.啮齿动物的标准立体定位头框,带有可调整的耳栏和咬合杆上的麻醉鼻锥。 B.大鼠以固定的耳条安装在立体框架中。 C.正确放置鼠标头在立体框架中,使得头部如图中红色虚线所示位于框架中央。 D.正确调整鼻锥和咬合杆高度,以确保头骨水平,如黄色虚线所示。


    4. 将动物手术体视显微镜下的手术区域集中在5x。

    5. 使用0.25%布比卡因诱导切口线块(不超过8 mg / kg)。
    6. 在头骨中线上做一个1厘米的切口(图2A),优于耳朵,低于眼睛。
    7. 通过将切口的每一侧从中线缩回来暴露头骨。
    8. 如果您在前囟点轻轻地推动颅骨板,并且缝线在轻微压力下移动时会变得明显,则现在可以轻松定位Bregma(图2B)。
    9. (0,0):(1,3),(1,-1),(4,3)和(4),在以下点标记优选的前肢感觉运动皮层(图2C)的立体定位坐标,-1)
    10. 在标记的坐标(图2D和2E),1-4mm侧面之间的矩形中稀释头骨; -1到+3毫米前囟,使用手术钻与牙髓电子马达系统设置为5000转的速度,配备了一个牙科手机配备和圆形硬质合金钻头设计用于切割。颅骨应该在3×4mm区域内均匀地变薄至0.1mm的厚度。
      注:颅骨变薄下的血管应清晰可见。如果您无法清楚地看到下面的脉管系统,可尝试在窗口上添加一滴无菌生理盐水以清除钻孔过程中产生的任何微粒的窗口。如果在添加一滴盐水后,基础血管不可见,则需要进一步稀释,继续稀释颅骨,直至血管变得可见。 


      图2.大鼠感觉运动皮层的光血栓形成A.中立切口暴露颅骨(用红色虚线表示)位于立体定位框架中的大鼠。 B.手术暴露的颅骨; C.使用手术显微镜(5倍放大率)相对于前囟(用黄色圆圈标记)建立的黑色圆圈标记的薄窗口角的立体坐标。 D.头骨的薄窗口(用黑框表示)在概述的坐标之间创建; C.在变薄的颅骨下的血管在放大5倍时变得可见; E.F。将激光靶向(用绿色圆圈表示)到薄窗口正下方的血管以诱导感觉运动皮质的缺血性梗塞,以5x放大倍数显示; G.

  4. 尾静脉注射光敏染料

    1. 按照30mg / kg的浓度计算注射所需的玫瑰红(在手术步骤A1之前制备)的体积:

    2. 用温水擦拭鼠尾以增加静脉的可见度(图3A和3B)。
    3. 用非优势手阻塞静脉(图3C)。
    4. 将针头滑入尾静脉,斜面朝上,针头平行于静脉(图3D和3E)。
    5. 血液闪光表明导管处于正确位置(图3F)。
    6. 将导管滑入静脉并取出针(图3G)。
    7. 确保有血液流出导管(图3F)。
    8. 用玫瑰红加上针。

    9. 注入孟加拉玫瑰红的计算体积(D1),速率为1毫升/分钟。
    10. 用0.2ml无菌盐水冲洗导管。
      注:玫瑰红可在注射后进入血管周围空间,导致尾静脉局部缺血并随后出现坏死性细胞死亡,盐水冲洗可预防此并发症。


      图3.大鼠尾静脉注射A.大鼠尾静脉的位置标有红色虚线。 B.将鼠尾旋转90°,使尾静脉变得可见。 C.用非优势手阻塞静脉。 D.使斜面朝上使导管针与尾静脉对齐。 E.将针平行于静脉。 F.插入针头,直到导管中有可见的血迹。 G.取出静脉留置导管的针头。 H.检查以确保血液流出导管以确保正确放置。导管现在准备好注射。 I.注射后取出导管。 J.施加压力使凝块形成。 K.确保尾巴看起来健康并且血流已经返回到尾尖。

  5. 诱导光血栓形成
    1. 戴上激光防护眼镜。
    2. 使用准直的绿色激光束(532nm,17mW;〜4.0mm光束直径)立即照亮体感皮层上的变薄的头骨15分钟以光激活玫瑰红(图2F和2G),遮蔽所有照亮的皮质脉管系统并诱发局灶性缺血性梗塞。
      注意:激光束位置的精确大小和位置将决定光凝血性中风的大小和位置。在尾静脉注射冲洗(步骤D3)之后立即打开激光束,因为假手术动物遵循所有的概述步骤,但是在15分钟曝光时间段内不打开激光(跳过步骤E2)。 /
    3. 在激光曝光器15分钟后,缝合颅骨并根据IACUC方案(0.05mg / kg皮下丁丙诺啡)给予止痛护理。
    4. 去除尾静脉导管。
    5. 皮下注射3毫升无菌生理盐水以防止脱水。
    6. 手术后恢复活动力时,观察动物与受伤皮层相关的前肢瘫痪和虚弱的迹象。
      注:手术后应立即在受伤肢体中出现明显缺陷。在大多数情况下,大鼠通过抬起并握紧受伤爪子避免使用中风前爪,而只使用未受伤前爪支撑体重,这一点很明显。

  6. 大鼠心脏灌注
    1. 准备灌注解决方案(4%福尔马林和含肝素盐水),并留出时间以达到正确的使用温度(方案2)。
      注:见Gage et al。 (2012年)的啮齿动物灌注详细协议。
    2. 根据美国兽医协会安乐死指南,以150 mg / kg腹腔注射戊巴比妥钠。
      注:对于500克大鼠,将计算待注射的Euthasol(400mg / ml戊巴比妥钠)的体积如下所示:

      500克大鼠的实例:将大鼠重量转化为千克:500克= 0.50千克。确定以毫克戊巴比妥钠注射的质量:0.500千克×150毫克/千克= 75毫克。确定Euthasol注射剂的体积(400 mg / ml):75 mg / 400 mg / ml = 0.188 ml 。
    3. 等待手术飞机到达,然后将动物背部固定在85°的灌注托盘上(图4A)。
    4. 定位剑突下面的剑突过程,在肋骨下面穿过覆盖物和腹壁进行横向切口。
    5. 使用钝头剪刀在膈肌上切开一个切口,沿着整个胸腔继续切口,露出胸膜腔。
    6. 通过肋骨两侧做矢状切口,确保不会损伤心脏或肺部。
    7. 提起并固定肋骨以显露冠状胸腔并释放胸腔附件的心脏(图4B)。
    8. 使用止血钳,握住心脏的顶端,并通过左心尖插入钝头16号针头,继续插入左心室(图4C)。
      注意:确保针头不会伸出主动脉弓很重要。
    9. 在右心房做一个小切口,释放液体压力,并以70-100 ml / min的速度启动启动的蠕动灌注泵(图4D)。
    10. 灌注250毫升37℃等渗盐水(细胞)肝素(5,000 IU / L)溶液(方案3)以清除动物的所有血液。
      注意:动物应该变得苍白,肝脏应该是白色的,并且在成功灌注时只有清澈的液体应该从右心房排出。
    11. 在4℃将灌注液转换为4%福尔马林溶液,再以70-100ml / min的速率灌注250ml溶液。
      注意:在4%福尔马林溶液输注后的几秒钟内,固定震颤应开始发生在整个大鼠身上,大鼠在灌注结束时应该非常僵硬。


      图4.经颅灌注大鼠A.大鼠置于85°灌注托盘上,虚线表示切口的位置。 B.手术暴露的冠状胸部,心脏突出。 C.通过心脏的左心尖插入钝头16号针头。 D.切入右心房释放液体压力并排出灌注液。

  7. 大鼠脑的集合
    1. 将颈部脊髓(C1)和头骨之间的动物斩首。

    2. 创建一个跨越颅骨的中线切口并移除所有皮肤以显露整个头骨。
    3. 将一个咬骨钳插入颈椎脱落后沿枕骨头滑动的空隙中。

    4. 切开枕骨颅骨,将骨头从后脑拉开,在另一侧重复。
    5. 沿中线在颅骨下方插入钝头剪刀,在中线剪下颅骨。

    6. 剥离颅骨的每一半远离大脑。

    7. 当大脑现在准备好从开放的颅骨腔体中移出时,可以夹住任何尖锐的边缘。
    8. 在额叶和嗅球之间插入一个小勺,释放大脑中的大脑。
    9. 运行大脑下的勺子,直到所有连接的组织血管被清除,并且大脑容易从开放的颅骨滑出。

  8. 组织固定,冷冻和冷冻切片
    1. 取出后立即将大脑置于50毫升4%福尔马林中,在4℃下放置过夜。
    2. 过夜固定后,用4°C 50毫升30%蔗糖溶液(配方4)替换4%福尔马林,并在4°C下离开组织3-4天,直到大脑不再浮于溶液中。

    3. 。从溶液中取出组织(组织会僵硬,白色)。

    4. 用一层OCT冷冻介质覆盖大脑
    5. 将大脑浸入冷却至-50℃的2-甲基丁烷溶液中。
    6. 让大脑淹没1分钟,以确保大脑整个冻结。
      注意:如果整个大脑长时间存放(超过2周),大脑应该涂上一层额外的OCT冷冻介质。
    7. 组织现在可以在-20°C长时间储存或立即冷冻切片。
      注意:如果计划对组织进行切片,我们不建议在-80°C下储存,因为组织切片时必须加热至-20°C。当我们在-80°C下储存后,将组织温热至最佳切片温度时,我们经历了组织的破裂和冻融线的出现。
    8. 一个低温恒温器应设置在-19°C和-22°C之间的温度,对于冠状脑切片,切割厚度为20μm。
    9. 切开整个大脑并将所有切片放在玻璃显微镜载玻片上。
      注意:如果进行免疫化学分析,建议使用Gold幻灯片,因为我们发现切片不能很好地粘附在未涂布的幻灯片上烘烤后。
    10. 幻灯片现在可以被复染或免疫标记。

  9. 中风病变的显微镜检查
    1. 在共聚焦显微镜上选择10倍显微镜物镜。
      注意:我们使用带Leica HCX PL APO L 10x 1.0NA水再现物镜和Leica LAS AF软件的Leica SP5共聚焦显微镜。
    2. 中风区域可以通过应用复染色,免疫标记的设置进行可视化,或者在某些情况下,可以使用FITC发射光谱中的自发荧光来有效定义中风边界。在未染色的组织中,可以使用光微分干涉对比或光DIC设置来确定中风边界。
    3. 使用显微镜软件设置参数来收集包含整个大脑切片的图像扫描图像(图5A)。
    4. 收集横跨梗塞的每个脑切片的图像(图5B)。


      图5.跨越缺血性梗塞的冠状切片A.大鼠脑的冠状切片,相对于前囟的侧向坐标,梗塞面积以红色显示。 B.对应于(A)中所示的每个冠状切片的行程区域,使用10倍放大倍数的共聚焦显微镜上的FITC发射光谱的光谱设置来可视化。改编自Wiersma 等,2017年。

  10. 缺血性病变的体积分析
    1. 通过分析横跨缺血性病变的每个冠状切片中的梗塞体积可以确定皮质的中风体积(图5)。
    2. 从导入到ImageJ中的每个冠状剖面导入收集的图像(程序I)(图6A)。
    3. 使用ImageJ中的测量功能,跟踪每个部分的梗塞,以确定每个切片中的中风的比表面积(图6B)。

    4. 将切片深度乘以表面积(20μm)以确定每个切片的损伤体积(图6C)。


      图6.一个冠状切片的梗塞体积计算示例A.使用10倍放大倍数的共聚焦显微镜上的FITC发射光谱的光谱设置显示切片中的梗塞清晰可见。 B.在ImageJ中追踪的梗塞区域用白色虚线表示。 C. ImageJ计算(B)所示的梗塞表面积,将其乘以切片厚度以提供切片中的每搏输出量。


    5. 每个切片跨越病变的梗塞体积可以相加,以确定总的每搏输出量。
    6. 预先确定排除标准并从研究中移除没有最小预定每搏量的动物是非常重要的(参见数据分析)。

  11. 局部缺血性梗塞的皮层映射
    1. 通过记录跨病变组织的每个冠状切片的中风边界的立体定位坐标,为每只动物创建显示诱发性中风的地形位置的皮层地图。
      对于横跨损伤的每个冠状切片,使用大鼠图谱来确定切片的y坐标(图7A)。
    2. 在建立的y坐标确定笔划边界的两个x坐标(图7A)。
    3. 绘制网格上的坐标(图7B)。
    4. 一旦绘制了所有的点,就可以将它们结合起来以创建中风区域的轮廓(图7C)。


      图7.找到中风皮层地形梗塞图的坐标A.冠状切片覆盖在大鼠图谱上以确定笔画的y坐标,然后笔划边界的x坐标为确定为切片(红色圆圈)。 B.边界坐标转移到网格上(红色圆圈)。重复此操作直至输入所有坐标,并在点之间绘制一条线以勾画出笔划边界。 C.网格可以叠加在皮层上以显示中风的位置。改编自Wiersma 等,2017年。

    5. 可以通过平均每个笔画边界的y坐标的立体定向x坐标来创建笔画区域的平均地形图,或者可以叠加笔画区域以显示梗塞大小和立体定位的恒定性。
    6. 应该预先确定中风的可接受位置,并且应该从研究中删除中风之外的动物(参见数据分析)。

数据分析

  1. 光血栓性中风在梗塞的体积和位置上高度一致。我们在雄性Sprague Dawley大鼠中进行n = 52次中风,发现平均中风大小为4.424mm 3,标准偏差为0.316,标准误差为0.0432(图8A)。
    我们建议排除每搏量在平均每搏量3个标准偏差之外的动物。
  2. 当所有52个脑卒中的地形图表示重叠时,很明显皮层脑卒中的位置非常稳定(图8B)。平均笔划地形图可以通过平均每个点的x和y坐标来创建。我们建议排除在任何位置距离平均坐标2毫米的动物。
  3. 当比较不同组之间的中风大小时,我们使用学生的不成对的双尾 t - 测试来确定组之间的中风体积是否显着不同。


    图8.脑卒中体积和位置的数据分析A.脑卒中体积为n = 52只动物,其平均值的长边中心线表示均值,标准误差线表示均值。 B.覆盖n = 52个笔划的地形皮层图。改编自Wiersma 等,2017年。

笔记

  1. 光血栓形成的中风模型是非常可重复的,因此在我们的研究中给予中风的52只动物中没有一只必须被排除,因为其中风的体积超过平均值3个标准差以上。在我们的研究中接受中风的所有动物显示由于缺血性损伤导致显着的行为缺陷。

食谱

  1. 玫瑰孟加拉解决方案
    1. 在一个小管里称100毫克的玫瑰红孟加拉
    2. 测量1毫升无菌生理盐水
      注意:所有的称重和测量步骤应该在最少的光线下完成。
    3. 加入生理盐水至孟加拉玫瑰,剧烈混合至所有玫瑰孟加拉溶液中。
      注:一旦玫瑰红在溶液中,它对光线非常敏感,应避免任何光线照射。
  2. 4%福尔马林溶液
    1. 测量300毫升等渗盐水

    2. 加入200毫升1:10福尔马林溶液
    3. 颠倒混合
    4. 冷至4°C
  3. 等渗盐水肝素(5,000 IU / L)溶液
    1. 测量500毫升等渗盐水
    2. 温水至37°C
    3. 添加2,500 IU肝素
      注意:肝素将在37°C变性超时,直到使用前才添加。
    4. 将肝素混入盐水中
  4. 30%蔗糖溶液
    1. 测量45毫升等渗盐水
    2. 加入15克D-蔗糖
    3. 大力混合
    4. 冷至4°C

致谢

这项工作得到了艾伯塔省创新健康解决方案,加拿大心脏和中风基金会,加拿大卫生研究院和加拿大自然科学和工程研究委员会的支持。来自加拿大创新基金会和艾伯塔省小型设备赠款的赠款用于获取共聚焦显微镜设备。该协议和数据来自以前的工作:Wiersma,A. M.,Fouad,K.和Winship,I. R.(2017)。 增强脊柱可塑性放大了康复训练的益处并改善了中风的康复 J Neurosci 37(45):10983-10997。作者没有利益冲突要申报。

参考

  1. 美国兽医协会安乐死指南(2013)(AVMA) 。
  2. Carmichael,S.T。(2005)。 啮齿动物的中风模型:大小,机制和目的 NeuroRx 2(3):396-409。
  3. Carmichael,S. T.,Archibeque,I.,Luke,L.,Nolan,T.,Momiy,J。和Li,S。(2005)。 中风后的生长相关基因表达:围梗死皮层中生长促进区的证据。 Exp Neurol 193(2):291-311。
  4. Dietrich,W. D.,Watson,B. D.,Busto,R.,Ginsberg,M. D.(1987)。皮层梗塞后的代谢可塑性:2-脱氧葡萄糖研究。在:Raichel,M. E.和Powers,W. J.(Eds)中。脑血管疾病。
  5. Gage,G.J.,Kipke,D.R。和Shain,W。(2012)。 啮齿动物全动物灌注固定 Vis Exp (65)。
  6. Ikeda,S.,Harada,K.,Ohwatashi,A.,Kamikawa,Y.,Yoshida,A。和Kawahira,K。(2013)。 光化学诱导脑梗塞的新型非人灵长类动物模型 PLoS一个 8(3):e60037。
  7. Que,M.,Schiene,K.,Witte,O.W。和Zilles,K。(1999)。 大鼠脑局灶性光致血栓形成损伤后广泛上调N-甲基-D-天冬氨酸受体。 Neurosci Lett 273(2):77-80。
  8. Sist,B.,Fouad,K.和Winship,I. R.(2014)。 围绕梗塞周围皮质的可塑性:脊柱向上调节结构可塑性,神经营养因子和炎症细胞因子在恢复过程中来自皮质卒中。 Neur Neur 252:47-56。
  9. Taylor,Z.J。和Shih,A.Y。(2013)。 有针对性地封闭小鼠皮层的单个软膜血管 Bio-Protocol e897 。
  10. Underly,R. G.和Shih,A. Y.(2017)。 体内双光子成像期间小鼠皮层毛细血管缺血的光致血栓诱导 Bio -protocol e2378。
  11. Watson,B.D.,Dietrich,W.D.,Busto,R.,Wachtel,M.S。和Ginsberg,M.D。(1985)。 通过光化学引发的血栓形成诱导可再现的脑梗塞 Ann Neurol 17(5):497-504。
  12. Wiersma,A. M.,Fouad,K.和Winship,I. R.(2017)。 增强脊柱可塑性放大了康复训练的益处并改善了中风的康复 J Neurosci 37(45):10983-10997。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Wiersma, A. M. and Winship, I. R. (2018). Induction of Photothrombotic Stroke in the Sensorimotor Cortex of Rats and Preparation of Tissue for Analysis of Stroke Volume and Topographical Cortical Localization of Ischemic Infarct. Bio-protocol 8(10): e2861. DOI: 10.21769/BioProtoc.2861.
  2. Wiersma, A. M., Fouad, K. and Winship, I. R. (2017). Enhancing spinal plasticity amplifies the benefits of rehabilitative training and improves recovery from stroke. J Neurosci 37(45): 10983-10997.
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