Name: Simultaneous Photothrombosis and Fiber Photometry to Induce and Monitor Ischemic Stroke in Behaving Mice (Video) | JoVE Journal
Uploaded: 2025-11-14T14:00:00.000+00:00
Duration: 7 min 49 s
Description: Learn to induce ischemic stroke in behaving mice while monitoring neuronal calcium dynamics using fiber photometry. This method enables simultaneous study of neurophysiological changes and behavioral outcomes.

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5 minutes

We thank the Cumming School of Medicine Optogenetics Platform for assistance in establishing this approach and the UNM Center for Brain Recovery &#38; Repair for additional training. Research by B.M. and A.K.J.B. reported in this publication was supported by the National Institute of General Medical Sciences of the National Institute of Health under award numbers P20GM109089 and P30GM159568 and operating grants to R.J.T. from the Canadian Institute for Health Research under award numbers PJT169174 and PJT168968. We also acknowledge SciDraw, a free repository of scientific drawings, and the artist Yunan Chen for the mouse cartoon used in Figure 1 (10.5281/zenodo.6645614).

The experimental procedure detailed in this article was done in accordance with the University of New Mexico Health Sciences Center Institutional Animal Care and Use Committee (IACUC protocol #24-201575-HSC) and in accordance with the Canadian Council on Animal Care guidelines and approved by the University of Calgary's Animal Care and Use Committees (protocol #AC21-0181). Thy1-GCaMP6f (C57BL/6J-Tg(Thy1-GCaMP6f)GP5.17Dkim/J, JAX 025393; P60-120) mice were group housed until the implant procedure, then single housed on a 12 h/12 h light/dark cycle. These mice were used for all experiments in this study. Room temperature and humidity were maintained at 21.4°C and 20%, respectively. Mice had ad libitum access to Purina laboratory chow and water. The reagents and the equipment used are listed in the Table of Materials.
1. Preoperative procedure
<ol>
	<li>Autoclave surgical instruments.</li>
	<li>Clean surfaces using 70% ethanol and place tools in a sterile field.</li>
	<li>Attach a sterile drill bit to the stereotaxic drill and mount it on the stereotaxic frame.</li>
	<li>Place the fiberoptic cannula into the stereotaxic fiberoptic cannula holder using needle drivers.</li>
	<li>Turn on surgical and recovery heating pads (37 °C).</li>
	<li>Place the recovery cage on the heating pad.</li>
	<li>Prepare 0.01 mL/g of buprenorphine stock (for a final dose of 0.01 mg/kg, i.e., 25 g mouse will receive 250 µL) in a 1 mL syringe with a 26 G ½" needle. This can be stored at room temperature and is not light sensitive.</li>
</ol>
2. Fiberoptic implant
<ol>
	<li>Weigh the mouse using a digital scale.</li>
	<li>Turn on oxygen flow towards the anesthetic induction chamber at 1 L/min.</li>
	<li>Place the mouse in the sealed chamber and induce anesthesia with 5% isoflurane (in O2) until an appropriate anesthetic depth is realized (absence of reflexes and ~ 1-2 Hz respiration).</li>
	<li>Switch anesthetic flow towards the nosecone, reducing to 1%-2% isoflurane in O2.</li>
	<li>Secure the mouse in the stereotaxic frame using the nose cone and earbars.</li>
	<li>Remove hair on the top of the head spanning from the anterior portion near the eyes and extending posterior to the nape of the neck using an electric razor (or hair removal product).</li>
	<li>Apply lubricating eye ointment using a cotton-tipped applicator to prevent drying during surgery.</li>
	<li>Clean the dorsal surface of the head with 3x serial applications of iodine and 70% ethanol.</li>
	<li>Verify absence of toe-pinch and blinking reflexes to ensure sufficient anesthetic depth.</li>
	<li>Inject 0.01 mg/kg buprenorphine solution i.p.</li>
	<li>Make a ~3 cm anterior-posterior (AP) incision to expose the skull.</li>
	<li>Using a cotton-tipped applicator, apply 5% hydrogen peroxide to remove periosteum from the dorsal section of the skull. 
	NOTE: It is crucial for the periosteum to be completely removed to ensure a secure bond of the dental cement to the skull and fiberoptic.</li>
	<li>Adjust the stereotaxic frame and ear bar to ensure that the skull is level in the dorsal-ventral (DV) and medial-lateral (ML) plane.</li>
	<li>Position the stereotaxic drill tip above bregma and adjust to the desired hippocampal coordinates (dorsal cornu ammonis (CA1), AP: - 2.0 mm, ML: - 1.3 mm).</li>
	<li>Lower stereotaxic drill to skull and drill a small burr hole, drilling in brief pulses followed by application of ice-cold saline to prevent heating of the skull and brain surface. 
	NOTE: Persistent bone drilling is known to cause heat and mechanical damage to the brain27.</li>
	<li>Replace the stereotaxic drill attachment with the fiberoptic cannula holder.</li>
	<li>Lower the optical fiber to make contact with the brain surface without penetrating and record DV coordinates.</li>
	<li>Slowly lower optical fiber DV -0.9 mm over at 0.1 mm per s to arrive immediately dorsal to the hippocampal CA1 region.</li>
	<li>Apply a quick air-dry dental cement using a precision applicator, covering the entirety of the exposed skull surface and the bottom ~ 1/3 of the fiberoptic cannula to secure in place. 
	NOTE: Ensure the application is not overlapping with soft tissue and skin to minimize the risk of lifting or removal of the cement cap. While uncommon, if these issues persist, scuffing the skull with a dental drill or using an etchant prior to dental cement application can improve cement adherence.</li>
	<li>Apply a second layer of UV-cured dental cement to support the implanted cannula, maintaining &gt;50% of the ferrule exposed.</li>
	<li>Suture the skin around the implanted fiberoptic cannula and cement cap.</li>
	<li>Turn off isoflurane, loosen the nosepiece, and place the mouse in a heated recovery cage.</li>
	<li>Monitor daily for post-surgical recovery (i.e., lethargy, signs of damage to the surgical area, redness and other signs of infection, bleeding, etc.), providing buprenorphine (0.01 mg/kg) i.p. for three days following surgery. Allow recovery for 7 days prior to handling.</li>
</ol>
3. Handling and habituation
NOTE : The goal of pre-experiment handling is to habituate the experimental mouse to handling, manipulation of the implanted fiber optic and ferrule, and tethering to the fiber optic patch cord.
<ol>
	<li>Day 1: Pick up and return the cage mouse repeatedly using a sweeping motion, holding the mouse in an open palm. Repeat this process for 5 min per mouse. 
	NOTE: To improve the initial habituation process, bedding can be picked up with the mouse.</li>
	<li>Day 2: With one hand, repeatedly pick up the mouse with a cupping motion and lightly cover with the opposite hand. Repeat this process for 5 min per mouse.</li>
	<li>Day 3: Pick up the mouse and cover lightly using one hand to secure the head. Using the opposite hand, touch the fiberoptic cannula (1-3 s intervals) and carefully return to the cage. Repeat this process for 5 min per mouse.</li>
	<li>Day 4: Pick up the mouse, secure the head, hold the fiberoptic cannula with small, subtle manipulation, and carefully place it down. Repeat this process for 5 min per mouse.</li>
	<li>Day 5: Pick up the mouse, secure the head, and attach a fiberoptic patch cord to the fiberoptic cannula. Place the mouse in a circular open field recording arena (5 min intervals) to acclimate the mouse to this environment and to the fiber-optic patch cord tether.</li>
</ol>
4. Equipment setup
NOTE: Combining fiber photometry of a green fluorescent biosensor with photothrombosis requires a setup allowing fluorescence excitation at 465 nm and an isosbestic wavelength (i.e., 405 nm) for the specific green fluorescent protein (GFP)-based biosensor excitation, along with photodetectors for emission collection (Figure 1A, B). Isosbestic excitation of fluorescent biosensors from light emitting diodes (LEDs) is commonly used for motion correction. In this setup, excitation and emission light are passed through a dichroic filter cube with an additional excitation port featuring a bandpass filter allowing laser excitation of Rose Bengal (i.e., 561 nm, Figure 1A,B). For long-term recordings, a fiber-optic-compatible rotary joint is recommended to minimize torque on the patch cord, thereby reducing impedance of animal behavior and signal noise (Figure 1A). While the representative recordings are from an open field arena (Figure 1C), this approach is amenable to most open-top behavioral arenas. Prior to beginning recordings, prepare a fresh Rose Bengal working solution each day. Dissolve Rose Bengal at 0.5 mg/mL in sterile saline (0.9 % NaCl) and sterile filter. Store at room temperature, covered with tin foil to protect from light until needed for injection.
<ol>
	<li>Turn on the 561 nm fiber-coupled laser. Adjust the 561 nm laser power to 6 mW (from 400 µm fiberoptic patchcord tip), evaluating with a digital handheld optical power meter. Switch to transistor-transistor logic (TTL) mode and attach a Bayonet Neill-Concelmant (BNC) cable to the input port to allow an external trigger.</li>
	<li>Turn on the fiber photometry console, LED driver(s), and detectors.</li>
	<li>Open the fiber photometry acquisition software on a compatible computer.</li>
	<li>Turn on the 465 nm LED to lock-in modulation (frequency-modulated) mode at maximum power. Set the intensity of the 465 nm LED to 30 µW (by adjusting the input voltage) from the tip of a 400 µm fiber optic patch cord, verifying the power using a digital handheld optical power meter.</li>
	<li>Turn on the 405 nm LED to lock-in modulation at a unique frequency and set to maximum power. Match the output voltage of the isosbestic LED (405 nm) to approximate the 465 nm LED voltage. 
	​NOTE: Frequency-modulation allows for simultaneous transmission of multiple LEDs at unique frequencies through the same optical fiber. Emission light is then demodulated during acquisition to differentiate the signal. Most systems offering frequency modulation have pre-set distinct frequencies for each LED. Assess the sensitivity of detection by repeatedly placing your finger tip at the fiber-optic patch cord output. If the LEDs and detectors are on, deflections in the signal will appear in your acquisition software. Wipe the patchcord tip with 70% ethanol once complete.</li>
	<li>Use digital input-output to set up a square-pulse for TTL input to the 561 nm laser with a 10 min delay to allow for baseline photometry and behavior recording (10 min delay, 8 min 'on' configuration23). Leave the laser turned 'on' to ensure that the external trigger is effective during recording.</li>
	<li>Plug the overhead USB camera into the computer.</li>
	<li>Use an additional digital input-output channel to set up the overhead behavioral camera to be externally triggered at a rate of 20 Hz.</li>
	<li>Adjust the camera so that the entire arena is visible in the field of view.</li>
</ol>
<img alt="Photometry diagram with LED driver, optical filter for brain excitation, emission analysis, behavior arena." class="xfigimg" src="/files/ftp_upload/69418/69418fig1v2.jpg" data-altupdatedat="1778558422000" data-alt="Figure 1"> 
Figure 1: Combined fiber photometry and photothrombosis experimental setup. (A) Frequency-modulated LED excitation light for GCaMP6f photometry (465 nm: Ca2+ dependent, 405 nm: isosbestic Ca2+ independent) is driven via the fiber photometry console and an LED driver to excite GCaMP6f. A digital output port from the fiber photometry console allows TTL-dependent control of the 561 nm fiber-coupled laser ('on' at 10 min, duration 8 min). LED and laser light are passed through the optical filter cube, such that excitation light of three wavelengths (405 nm, 465 nm, and 561 nm) is directed through the fiber optic tether and the implanted fiberoptic cannula to reach the mouse. An optional rotary joint can be included in the light path to reduce movement artifact for prolonged recordings. Emission light returns through the fiber back through the optics and a bandpass filter for green fluorescence (500-540 nm) prior to being directed towards the photodetector. Here, the signal is digitized and demodulated to separate the input from the 405 nm and 465 nm LEDs. This signal is then sent to the computer for signal processing (i.e., correction for photobleaching and animal movement, normalization) and analysis (i.e., peak detection, TD amplitude, onset, and maximum derivative). The behavioral camera is controlled via transistor-transistor logic (TTL) input from the fiber photometry console, acquiring frames (20 Hz or 30 Hz) synchronized with fiber photometry recordings. Data is sent to the computer for manual (behavioral state) or automated analyses (pose estimation, locomotion, etc.). (B) Schematic of fiberoptic implant dorsal to hippocampal CA1, highlighting excitation wavelengths of LEDs and 561 nm laser and emission of green fluorescent biosensor, GCaMP6f. (C) Example of a tethered mouse in the behavioral arena. <a href="https://www.jove.com/files/ftp_upload/69418/69418fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Awake behaving photothrombosis with fiber photometry
<ol>
	<li>Weigh the mouse using a digital scale.</li>
	<li>Scruff the mouse and wipe the fiberoptic cannula of any debris using a lint-free wipe sprayed with 70% ethanol.
	<ol>
		<li>For stroke mice, administer 0.5% Rose Bengal (5 mg/mL) to the mouse (50 mg/kg) via i.p. injection.</li>
		<li>For sham mice, substitute Rose Bengal for vehicle (saline) i.p. at an equivalent volume per weight (0.01 mL/g).</li>
	</ol>
	</li>
	<li>While the mouse is scruffed, connect the fiber-optic patch cord to the implanted fiber-optic ferrule by gently twisting the sleeve while applying consistent pressure. 
	NOTE: The diameter and numerical aperture of the fiberoptic in the patch cord and the cannula should match, and the outer diameter of the patch cord tip and the implanted fiberoptic ferrule must match for efficient light delivery. Ensure the end of the patch cord and ferrule are in direct contact to guarantee efficient light transmission. Evidence of light leak from a loose connection can be visually confirmed once the recording has started. If present and identified within the first 2 min of the recording, stop the recording, adjust the connection, and restart.</li>
	<li>Place the animal into the recording arena.</li>
	<li>Initiate the camera, followed by hitting 'record' on photometry acquisition software to ensure time-locked acquisition. 
	NOTE: Depending on the photometry setup, these features may be initiated simultaneously using digital outputs and software integration. Assess the initial minute of recording for evidence of a successful recording (i.e., presence of photobleaching curve, transients visible in the 465 nm channel that are absent in the isosbestic). If these are absent, stop the recording and see the troubleshooting suggestions in the representative results (Figure 2).</li>
	<li>After 40 min, end recording in the photometry acquisition software. Turn off the camera and the 561 nm laser.</li>
	<li>Pick up the mouse, secure the head in a similar fashion to the fiberoptic attachment protocol, and remove the fiberoptic patch cord with a slow twisting motion.</li>
</ol>
6. Data integration and analysis
NOTE: For data analysis, export photometry data (.csv) and behavior video (.avi) onto an analysis computer. An appropriate photometry analysis package will include: photobleaching correction (based on exponential fit) and movement correction (based on normalization to the isosbestic channel). Several open-source and proprietary analysis options are also available. Photometry analysis is not the focus of this protocol, given the diversity of recording setups available, and is highly dependent on the research question; however, a modifiable analysis package and instructions for use can be found here: https://doi.org/10.5281/zenodo.8303055. Comprehensive behavior analysis is constrained by the targeted brain structures(s), the arena, and the camera resolution and orientation (i.e., digging will not be scorable in an open field; however, it applies to home-cage; sleeping vs. stationary is difficult to identify with a low-resolution overhead camera). 
<ol>
	<li>Manually score behavioral state using an Excel-based Macro (developed here28). 
	NOTE: In the open field with "bird's eye view" camera (see Figure 1), behaviors were manually scored as stationary (the mouse is still), ambulating (the mouse is moving in the arena), rearing (the mouse is sitting back on its hindlegs touching the walls of the arena with its forepaws), or grooming (repetitive sweeping motions of paws focused towards its head), which were then converted to a Gantt plot (Figure 2).
	<ol>
		<li>Initiate Macro, with desired 'starting position' (i.e., the state that the mouse is in at the onset of the behavior recording) simultaneously with pressing 'play' in each behavior video.</li>
		<li>Use the keyboard to score transitions in the behavior state.</li>
		<li>Once the video is completed, hit stop on the Macro to generate an Excel output.</li>
		<li>Generate a Gantt plot based on behavioral state over time that can be manually aligned with the respective photometry trace (see Figure 2).</li>
	</ol>
	</li>
</ol>
<img alt="Neuronal Ca²⁺ imaging diagram with fluorescent biosensor detection and TTC stain infarct evaluation." class="xfigimg" src="/files/ftp_upload/69418/69418fig2v2.jpg" data-altupdatedat="1778558422000" data-alt="Figure 2"> 
Figure 2: Onset of ischemic terminal depolarization (TD) in the hippocampus coincides with ambulation. (A) Representative photometry (neuronal GCaMP6f) and behavioral recording (Gantt plot) during induction of ischemic hippocampal stroke in an awake behaving Thy1-GCaMP6f mouse. Representative 40 min recording (10 min baseline followed by 8 min 561 nm illumination (6 mW) and 22 min follow-up). Behavioral categories for Gantt plot: stationary, grey; ambulating, green; grooming, orange; rearing, magenta. (B) Representative evidence for biosensor expression at the onset of recording as a slow exponential decay related to photobleaching that is absent in GCaMP6f-negative mice. (C) An optimal 30 s window of baseline photometry recording demonstrating GCaMP6f transients paired with two common issues that are identified in a photometry recording, absence of GCaMP6f dynamics indicating misplacement of fiber, or absence of biosensor expression and signal dropout caused by a loose ferrule-patch cord connection. (D) An example of the transition to photothrombotic illumination highlights efficient light transmission relative to the reflectance observed when a gap exists between the fiberoptic cannula and the patch cord. (E) Infarct evaluation using 2,3,5-triphenyltetrazolium chloride (TTC) highlights an optimal unilateral hippocampal infarct (white) and live (red) brain tissue at 48 h post-ischemia induction, as well as the absence of this infarct following sham illumination. Several suboptimal histological results, including off-target or unstable fiber implant as well as over- or under-illumination, are also revealed. Scale bar: 2 mm. <a href="https://www.jove.com/files/ftp_upload/69418/69418fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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<li>Boyce, A. K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contralesional+hippocampal+spreading+depolarization+promotes+functional+recovery+after+stroke%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Contralesional hippocampal spreading depolarization promotes functional recovery after stroke.</a> Nat Commun. 16 (1), 3428(2025).</li>
<li>Simpson, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((action%21+A+primer+on+in+vivo+fiber+photometry%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">action! A primer on in vivo fiber photometry.</a> Neuron. 112 (5), 718-739 (2024).</li>
<li>Szabo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hippocampal+stroke%5BTitle%5D)+AND+%22Hippocampus+Clin+Neurosci%22%5BJournal%5D)">Hippocampal stroke.</a> Hippocampus Clin Neurosci. 34, 150-156 (2014).</li>
<li>Blanco-Suárez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photothrombotic+model+to+create+an+infarct+in+the+hippocampus%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Photothrombotic model to create an infarct in the hippocampus.</a> Methods Mol Biol. 2616, 29-38 (2023).</li>
<li>Shoffstall, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potential+for+thermal+damage+to+the+blood-brain+barrier+during+craniotomy%3A+Implications+for+intracortical+recording+microelectrodes%5BTitle%5D)+AND+%22J+Neural+Eng%22%5BJournal%5D)">Potential for thermal damage to the blood-brain barrier during craniotomy: Implications for intracortical recording microelectrodes.</a> J Neural Eng. 15 (3), 034001(2018).</li>
<li>Füzesi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypothalamic+CRH+neurons+orchestrate+complex+behaviours+after+stress%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Hypothalamic CRH neurons orchestrate complex behaviours after stress.</a> Nat Commun. 7, 11937(2016).</li>
<li>Oitzl, M. S., Huston, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electroencephalographic+spreading+depression+and+concomitant+behavioral+changes+induced+by+intrahippocampal+injections+of+ACTHl_24+and+D-Ala+2-Met-Enkephalinamide+in+the+rat%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Electroencephalographic spreading depression and concomitant behavioral changes induced by intrahippocampal injections of ACTHl_24 and D-Ala 2-Met-Enkephalinamide in the rat.</a> Brain Res. 308, 33-42 (1984).</li>
<li>Mitlasóczki, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hippocampal+spreading+depolarization+as+a+driver+of+postictal+ambulation%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">Hippocampal spreading depolarization as a driver of postictal ambulation.</a> Sci Transl Med. 17, adv3260(2025).</li>
<li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high+signal-to-noise+Ca2%2B+probe+composed+of+a+single+green+fluorescent+protein%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein.</a> Nat Biotechnol. 19, 137-141 (2001).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fast+and+sensitive+GCaMP+calcium+indicators+for+imaging+neural+populations%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Fast and sensitive GCaMP calcium indicators for imaging neural populations.</a> Nature. 615 (7954), 884-891 (2023).</li>
<li>Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+impact+of+reporter+kinetics+on+the+interpretation+of+data+gathered+with+fluorescent+reporters%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">The impact of reporter kinetics on the interpretation of data gathered with fluorescent reporters.</a> bioRxiv. , (2019).</li>
<li>Mathis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DeepLabCut%3A+Markerless+pose+estimation+of+user-defined+body+parts+with+deep+learning%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">DeepLabCut: Markerless pose estimation of user-defined body parts with deep learning.</a> Nat Neurosci. 21 (9), 1281-1289 (2018).</li>
<li>Clark, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Artery+targeted+photothrombosis+widens+the+vascular+penumbra%2C+instigates+peri-infarct+neovascularization+and+models+forelimb+impairments%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Artery targeted photothrombosis widens the vascular penumbra, instigates peri-infarct neovascularization and models forelimb impairments.</a> Sci Rep. 9 (1), 2323(2019).</li>
<li>Shih, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+smallest+stroke%3A+Occlusion+of+one+penetrating+vessel+leads+to+infarction+and+a+cognitive+deficit%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">The smallest stroke: Occlusion of one penetrating vessel leads to infarction and a cognitive deficit.</a> Nat Neurosci. 16 (1), 55-63 (2013).</li>
<li>Peña-Martínez, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacological+modulation+of+neutrophil+extracellular+traps+reverses+thrombotic+stroke+tPA+resistance%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Pharmacological modulation of neutrophil extracellular traps reverses thrombotic stroke tPA resistance.</a> Stroke. 50 (11), 3228-3237 (2019).</li>
<li>Sun, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+murine+photothrombotic+stroke+model+with+an+increased+fibrin+content+and+improved+responses+to+tPA-lytic+treatment%5BTitle%5D)+AND+%22Blood+Adv%22%5BJournal%5D)">A murine photothrombotic stroke model with an increased fibrin content and improved responses to tPA-lytic treatment.</a> Blood Adv. 4 (7), 1222-1231 (2020).</li>
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An all-optical technique is utilized to initiate ischemic stroke in freely behaving mice, allowing for the simultaneous collection of neurophysiological data using fiber photometry of a green fluorescent biosensor (i.e., neuronal Ca2+ via Thy1-GCaMP6f23). Several &#34;plug-and-play&#34; commercially available fiber photometry systems now exist, making photometry accessible24. These customizable and basic fiber photometry systems for recording green fluorescent biosensors are relatively affordable. Adapting a basic system to allow for simultaneous photothrombosis requires a relatively low-cost and minor modification of the filter cube (allowing an extra excitation line at 561 nm) and the integration of a fiber-coupled laser. Here, transgenic Thy1-GCaMP6f mice were used; however, this approach is amenable to transgenic or viral expression of any green fluorescent indicator. Photometry surgeries are less invasive than other forms of intravital microscopy (miniaturized microscopy, multiphoton, etc.), particularly in the context of deeper brain structures that require resection of overlying tissue and/or rely on gradient index (GRIN) lens implantation. Lastly, raw data from fiber photometry experiments is smaller in size and less complex than in vivo electrophysiology and microscopy. While data processing and analyses require an understanding of the caveats (discussed below), these processes require significantly less computing power and are more permissive to sharing across collaborations. In this protocol, the focus is on neuronal Ca2+ dynamics and TD in the stroke core; however, this technique can be paired with additional fiberoptic cannulas or electrodes to understand spreading features of the ischemic TD and/or neurophysiological changes in brain tissue remote to ischemia, as was done in a preceding study23.
It is important to recognize that fluorescent biosensors are sensitive to changes in their molecular environment, including pH and hemodynamics, which are particularly significant in pathological contexts, as well as photobleaching and animal motion. These factors are important considerations when choosing a biosensor and evaluating the data. Photobleaching can be corrected by fitting an exponential to the trace. Typically, photobleaching stabilizes after ~5 min of recording; thus, a 10 min baseline recording is recommended so that the latter half of the recording is free from exponential decay of the photometry signal. Motion correction can be accomplished via normalization to the isosbestic output on the biosensor. Aside from photobleaching and motion correction, the physiological impacts of the stroke core require careful consideration. Following vessel occlusion, ischemic tissue sees an abrupt decrease in blood flow and tissue oxygenation, along with a local decrease in extracellular pH (to ~pH 6.5)2,5. These processes can be further exacerbated either permanently or transiently by spreading depolarization5. The dynamic range of GCaMP decreases with tissue acidification31,32 thus it is possible that coincident changes in amplitude of neuronal Ca2+ and GCaMP are mildly decoupled in the stroke core relative to na&#239;ve tissue. Given the pan-cellular sustained depolarization that occurs in a permanent occlusion model like photothrombosis, the difference in kinetics of fluorescent reporters relative to their ligands is less of a concern than when recording behavior-associated biosensor fluctuations in na&#239;ve tissue; however, this should still be considered (see a detailed discussion here33).
Using this approach, neurophysiological dynamics in ischemic tissue can be directly tied to changes in behavior. Here is an example of using manual behavior state tracking via a categorical analysis platform (developed in28) to identify increased ambulation following the onset of ischemia-evoked hippocampal TD. In a recent study, the amplitude of ischemia-evoked hippocampal TD correlated with lesion size at 48 h post-stroke and was inversely correlated with post-stroke performance in contextual fear conditioning23, suggesting ischemia-evoked TD can be a real-time proxy for stroke severity. This permits longitudinal within-animal study of post-stroke recovery. In a previous study23, behavior state outputs were integrated with the photometry signal using a MATLAB script that can be found here (https://doi.org/10.5281/zenodo.8303055), which generates a behavioral average for a defined epoch before and after the TD. If interested in pose estimation and locomotion tracking, a specific DeepLabCut (DLC)-based analysis algorithm can be developed to track behaviors of interest in each video; however, this will be specific to the experimental design, ischemic brain structure, as well as the method and resolution of tracking. In the aforementioned previous study, A MATLAB script to align DLC outputs tracking of center-of-mass from an overhead camera with photometry is available here (https://doi.org/10.5281/zenodo.8303055). This code can be modified to track unique &#8220;body parts&#8221; in an open field over time relative to fluctuations in the photometry signal.
There are several considerations to remember when interpreting data from these awake-behaving experiments. Given the localized nature of photothrombotic illumination, penumbral tissue is often short-lived, minimal, or even absent35. This is particularly the case when photothrombosis is used to non-specifically target the neocortex. In some cases, focused lasers have been used to specifically target large surface or penetrating vessels on the neocortex for occlusion36. Here, the likelihood of penumbra is increased, in particular when arteries are targeted35. Using a fiberoptic tether and an optical fiber placed directly over the anterior branch of the distal MCA, a group was able to generate MCA occlusion in freely behaving mice; however, this group did not collect neurophysiological information during occlusion17. In the case of optical fiber-based deeper brain strokes, reduced optical access impairs the ability to generate a single vessel occlusion. Rose Bengal photothrombosis is also resistant to reperfusion and responds poorly to thrombolytic agents, meaning that the penumbra that does form has limited opportunity for recovery. This is likely due to the presence of platelet-rich, fibrin-poor clots and may be ameliorated by including thrombin in an admixture with Rose Bengal dye injection37,38 or by illuminating during a critical time window with 355 nm laser light39.
This all-optical approach to induce ischemic stroke in the hippocampus of a behaving mouse enables simultaneous monitoring of neuronal Ca2+ in the emerging stroke core. Importantly, fiberoptic cannulas allow for the flexibility to target any brain region; therefore, this model can be adapted to generate ischemic strokes in any structure. Photothrombosis via a chronic fiberoptic implant allows scalable targeted ischemic stroke induction in the absence of the neuroprotective effects of anesthesia. A fiberoptic patch cord tether enables the analysis of unencumbered peri-stroke behavior, allowing for a better understanding of region-specific symptomology. While initially established using the genetically encoded Ca2+ indicator GCaMP6f, simultaneous photothrombosis and fiber photometry is amenable to use with any green fluorescent biosensor.

Approximately one in four people who reach adulthood will suffer a stroke in their lifetime, where ischemic stroke represents the vast majority1. Ischemic stroke is triggered by a blockage of blood flow in the cerebral vasculature (for a detailed review, see2). Following vessel occlusion and the subsequent perfusion deficit, metabolic substrates, including glucose and oxygen, rapidly decline. This generates a metabolic crisis in the tissue supplied by the occluded vessel, and energy-dependent processes within brain cells begin to fail. Importantly, maintenance of the negative resting membrane potential of neurons is an energy-dependent process, where Na+ and K+ are pumped against their concentration gradient by Na+/K+ ATPase3. In the absence of adenosine triphosphate (ATP), there is a near-complete breakdown, leading to elevated intracellular Na+ and Ca2+ and extracellular K+, as well as release of neurotransmitters4. Nearby cells are subsequently depolarized by the elevated extracellular K+ and glutamate, triggering a slowly propagating wave of pan-cellular depolarization followed by a varying period of electrical depression, as the cells attempt to re-establish their membrane potential5. As this is an energy-dependent process, the length of the depolarization and subsequent depression are inversely related to the perfusion status of the tissue. Sustained depolarization pushes brain cells past a &#39;commitment point&#39; towards death, and thus this spreading depolarization (SD) becomes terminal and is deemed the terminal spreading depolarization (TSD)6 if reperfusion is absent or insufficient. There is no objective duration to reach the commitment point, as it will be impacted by the perfusion status, model, tissue, and cellular context, as well as the experimental method used to identify cell death. In the stroke core, this TSD is often called the anoxic depolarization, due to its onset occurring in response to metabolic crisis following vessel occlusion3. In susceptible and hypoperfused tissue adjacent to the stroke core (penumbra), SDs can also be generated in response to supply-demand mismatch, where local energy requirements are not met by adequate perfusion7.
To understand the mechanism underlying anoxic TSD, preclinical murine models have been developed to reliably induce stroke in the lab. Several of these models rely on complex surgical procedures and thus are performed under anesthesia, including cerebral artery occlusions with externally applied clips or sutures, intraluminal filament8, or intravenous occluding nanoparticles (i.e., exogenous thrombin, nanobeads, or magnetic nanoparticles)9,10,11,12. In 1985, Watson et al. developed the photothrombotic stroke model13. Here, the photo-activatable dye Rose Bengal is administered intraperitoneally or intravenously so that it circulates through the bloodstream. Next, focused green laser light (commonly ~530-560 nm) is used to illuminate a volume of tissue or a single vessel to activate the photosensitizer Rose Bengal, which generates reactive oxygen species, damaging the local endothelium and initiating the clotting cascade to eventually generate vessel occlusion14,15. While this is still primarily done under anesthesia, some groups perform photothrombosis in awake mice, commonly targeting neocortical surface vessels in a head-fixed preparation16.
There has been a push to move towards stroke induction in awake mice for several reasons. First, isoflurane anesthesia is well-known to be neuroprotectant during stroke17&#160;via several converging mechanisms, including competing for the glycine site to antagonize the N-methyl-D-aspartate (NMDA) glutamate receptors18, inhibiting SD19, augmenting neurotransmission20, impacting temperature regulation21, and altering blood flow22. Importantly, isoflurane can mask potential beneficial effects of interventions17. Further, induction of stroke23 in a behaving animal allows understanding and interrogation of the symptomology of stroke, which is otherwise impossible in an anesthetized animal.
A combination of existing technologies was utilized to create an all-optical method for inducing ischemia and monitoring neuronal Ca2+ in the emerging stroke core of awake and freely behaving mice by delivering and collecting light through a single optical fiber23. An all-optical approach allows for simultaneous interrogation of behavior and neurophysiology in awake animals via a flexible fiberoptic patch cord tether and fiber photometry. Fiber photometry is an optical approach for exciting and recording changes in bulk tissue fluorescence of a biosensor expressed in vivo that is used as a proxy for cellular or protein activity, neurotransmitter release, ion flux, etc. (for a comprehensive review, see24). Here, excitation light passes through an implanted fiberoptic cannula, exciting fluorescent molecular biosensors expressed&#160;in the tissue volume below the fiber tip. Fluorescence returns&#160;through the same optical fiber and arrives at a photodetector or camera, then&#160;it is converted to a digital signal that serves as a proxy for the amount of ligand-bound biosensor (i.e., intracellular Ca2+ and GCaMP). The specifications of the optical fiber allow the collection of light from tissue volumes ranging from 50-400 &#181;m beneath the fiber tip. This &#39;bulk&#39; signal thereby represents changes in biosensor fluorescence in localized groups of cells and does not represent individual cellular resolution. Given that most of these biosensors are genetically encoded, fiber photometry permits repetitive recording of specific cellular populations across many sessions spanning days and months. The lightweight and low-profile implant allows mice to behave relatively unencumbered compared to many other neurophysiological approaches. While fiber photometry has gained significant popularity among neuroscientists, very few groups have used it for recording SD or pathological neuronal activity, in general. Photothrombosis has been used in freely behaving mice17, yet it was not paired with fiber photometry until a recent publication from an associated author23. Here, a protocol is presented to simultaneously record changes in neuronal activity using fiber photometry before, during, and after the terminal depolarization in ischemic stroke initiated with photothrombosis, and how this relates to mouse behavior. Demonstrated here is its utility for triggering deep brain strokes by initiating unilateral hippocampal ischemia (see also23), which causes amnestic symptoms in humans25. While hippocampal strokes are the focus of this protocol, coordinates for fiberoptic implantation can be adjusted to evoke and record ischemic stroke in any brain region. Importantly, implanted optical fibers have been used to initiate photothrombosis in the hippocampus26; however, this protocol demonstrates the utility of combining photothrombosis with fiber photometry recordings in ischemic tissue. Taken together, an all-optical method is presented combining green fluorescent biosensor (i.e., GCaMP6f) photometry (465/405 nm) to understand neurophysiology in the emerging stroke core with photothrombotic stroke (561 nm) through a single fiberoptic cannula, allowing for scalable ischemia generation in behaving mice. While this paper utilizes transgenic Thy1-GCaMP6f mice with sparse expression of the genetically encoded Ca2+ indicator in pyramidal neurons, this protocol can be adapted for any green fluorescent biosensor in any brain cell type.

<table><tbody><tr><td>#10 Scalpel blade</td><td>World Precision Instruments</td><td>500239</td><td>Scalpel blade</td></tr><tr><td>100 mW 561 nm DPSS fiber-coupled laser (&amp;lt; 3% stability)&amp;nbsp;</td><td>UltraLasers</td><td>MGL-FN-561-100</td><td></td></tr><tr><td>2,3,5-triphenyltetrazolium chloride</td><td>Sigma</td><td>T8877-10G</td><td></td></tr><tr><td>Assisted 1x1 Fiber-optic Rotary Joint, 400 um NA0.57 fibre, 1m input, optimized for fiber photometry</td><td>Doric</td><td>AFRJ_1x1_PT_400-0.57</td><td></td></tr><tr><td>brain block (mouse coronal)</td><td>WPI</td><td>RBMA-200C</td><td></td></tr><tr><td>Data Analysis Software, 1 licence</td><td>Doric</td><td>danse 1-year subscription</td><td></td></tr><tr><td>Doric Neuroscience Studio</td><td>Doric</td><td></td><td>Fiber Photometry Acquisition Software</td></tr><tr><td>Fiber Photometry Console</td><td>Doric</td><td>FPC (United States)</td><td></td></tr><tr><td>Fiberoptic cannula mative sleeve (2.5 mm)</td><td>Doric</td><td>SLEEVE_ZR_2.5</td><td></td></tr><tr><td>Fluorescence Mini Cube - 5 ports with 1 integrated photodetector, 2 integrated LEDs and 1 port for red opsin</td><td>Doric</td><td>ilFMC5-G3_IE(400-410)_&lt;br/&gt; E(460-490)_F(500-540)_&lt;br/&gt; O*(555-570)_S</td><td>Fluorescence mini-filter cube optics with integrated LEDs, LED drivers, and detectors. It is possible to purchase each of these components separately with the trade-off for more flexibility in illumination and detection with decreased efficiency</td></tr><tr><td>GC Unifast LC A2, 1:2 Package - Light-Cure Temporary Crown and Bridge Material, 1 - #338002</td><td>US Dental Depot</td><td>421373</td><td>LED curing dental cement (protects fiber from physical damage)</td></tr><tr><td>LIGHT-S-5 B GREEN Dental Wireless LED Curing Light Unit - 2000 mW</td><td>US Dental Depot</td><td>16595</td><td>Cure dental cement</td></tr><tr><td>Metabond kit</td><td>Fisher Scientific</td><td>NC0764473</td><td>Secure fiberoptic cannula on to skull</td></tr><tr><td>Mono fiberoptic cannula</td><td>Doric</td><td>MFC_400/430-0.48_1.5mm_&lt;br/&gt;ZF2.5(G)_FLT</td><td>1.5 mm fiberoptic cannula with 400 mm inner diameter (NA 0.48)</td></tr><tr><td>Mono Fiberoptic Patchcord - Low AutoFluorescence</td><td>Doric</td><td>MFP_400/430/1100-0.57_1m_CM3-MF2.5_LAF</td><td></td></tr><tr><td>Mono Fiberoptic Patchcord (connecting 561 nm laser to mini-cube)</td><td></td><td>1 MFP_200/220/900-0.22_0.3m_FC-FC</td><td></td></tr><tr><td>Mouse Dissecting Kit</td><td>World Precision Instruments</td><td>MOUSEKIT</td><td>Kit suitable for minor cranial surgeries</td></tr><tr><td>Power meter</td><td>Thor labs</td><td>PM100D</td><td></td></tr><tr><td>Rose Bengal</td><td>Sigma</td><td>330000-5G</td><td></td></tr><tr><td>Scalpel handle&amp;nbsp;</td><td>World Precision Instruments</td><td>500236-G</td><td>Scalpel handle for #10 scalpel blade</td></tr><tr><td>Sensor/Fiber optic adaptor</td><td>Thor labs</td><td>S130C</td><td></td></tr><tr><td>Stereotaxic cannula holder (2.5 mm)</td><td>Doric</td><td>SCH_2.5</td><td>Cannula holder for implantation during stereotaxic surgery</td></tr><tr><td>Stereotaxic Drill Micromotor Kit</td><td>Foredom</td><td>K.1070</td><td>Drill for stereotaxic frame</td></tr><tr><td>Stereotaxic in-line adaptor</td><td>Doric</td><td>SIA_7.9</td><td></td></tr><tr><td>Steri-lite Glass Bead Sterilizer</td><td>World Precision Instruments</td><td>ST5193</td><td>Bead sterilizer</td></tr><tr><td>USB3.0 Behavior Tracking Camera system Color</td><td>Doric</td><td>BTC_USB3.0_CO</td><td></td></tr></tbody></table>

simultaneous photothrombosis and fiber photometry to induce and monitor ischemic stroke in behaving mice

Simultaneous Photothrombosis and Fiber Photometry to Induce and Monitor Ischemic Stroke in Behaving Mice

Stroke is a leading cause of death and disability across the globe. During ischemic stroke, an obstructed cerebral blood vessel causes focal metabolic failure, neuronal membrane potential breakdown, and dramatic neuronal Ca2+ influx as part of the anoxic depolarization. Without timely reperfusion, prolonged depolarization in the anoxic core is terminal. The dysregulated metabolic environment, aberrant cellular activity, and subsequent tissue infarction associated with ischemic stroke cause both acute and lasting symptoms. This study presents an all-optical method for inducing ischemia and monitoring neuronal Ca2+ in the emerging stroke core of awake and freely behaving mice by delivering and collecting light through a single optical fiber. Here, stereotaxic fiberoptic cannula implantation and sufficient recovery time in advance of experimentation permit flexibility in generating a focal stroke in a brain region of interest, without the need for anesthesia. Mice are then tethered to a fiberoptic patch cord for scalable focal ischemia induction via Rose Bengal photothrombosis. Strokes are performed in transgenic Thy1-GCaMP6f mice, featuring sparse expression of the genetically encoded Ca2+ biosensor GCaMP6f in pyramidal neurons. To record neuronal Ca2+ dynamics in the emerging stroke core, the same optical fiber is utilized for simultaneous GCaMP photometry. This murine stroke model is flexible for any brain region of interest, allowing scalability with laser illumination power and duration, as well as simultaneous behavioral assessment, and is amenable to any green fluorescent biosensor. Together, combining photothrombosis and fiber photometry in behaving mice enables a precise and region-specific investigation of symptomology during acute stroke, in the absence of the neuroprotective effects of anesthesia.

Following illumination of naïve brain tissue of a mouse pre-injected with Rose Bengal, a dramatic increase is observed in GCaMP6f fluorescence in the emerging stroke core, corresponding to massive and sustained Ca2+ influx in pyramidal neurons and neighboring cells during the anoxic depolarization (Figure 2A). Given the persistent nature of this sustained increase in neuronal Ca2+ and the eventual infarct that forms under the fiberoptic tip, this is a bona fide terminal depolarization (TD). Importantly, while this persistent influx of Ca2+ shares features with the terminal spreading depolarization, the use of a single fiber does not allow for the detection of spread, so it will be referred to as the TD. In sham mice, where Rose Bengal is substituted for its vehicle (saline) alone, there is no change in neuronal Ca2+ and no evidence of an infarct when tissue is later processed using TTC (Figure 2).
There are several indicators of an unsuccessful recording, many of which have the potential to be ameliorated during the recording session. First, in instances where there is an absence of a biosensor (i.e., incorrect genotype, misplaced fiber), there will be little to no photobleaching at the beginning of the photometry recording (Figure 2B). This can be ameliorated by genotyping, when using a transgenic mouse, or carefully delivering viral injection followed by fiberoptic cannula implantation during the same stereotaxic surgery, when using viral biosensor expression. While an exponential fit can correct much of the photobleaching curve, it is suggested to remove the initial 5 min of the recording from the analysis to ensure the analysis has a stable photometry signal23. An optimal hippocampal neuronal GCaMP6f recording in Thy1-GCaMP6f mice has a stable baseline with spontaneous transients (~5-10 a.u.). The absence of these transients could suggest the absence of biosensor expression or a misplacement of the fiberoptic during stereotaxic surgery (Figure 2C). This could also be explained by a loose connection between the tip of the fiberoptic patch cord and the implanted ferrule. A clear indication of a loose or unstable connection can be observed in large, sharp changes in the baseline signal during movement. Ensuring that the connector sleeve overlaps equally with both the patch cord and the ferrule is important for a stable connection. An additional indicator of a loose connection will appear during photothrombotic illumination (Figure 2D), where large amplitude reflectance noise can be observed in the recording. Lastly, a live-dead stain like 2,3,5-triphenyltetrazolium chloride (TTC) can be used to establish appropriate illumination times that generate a desired infarct size in the correct brain region (Figure 2E). TTC is a colorless substrate that is converted to a red product in living cells; thus, the infarct is visible post-stroke as colorless tissue. Appropriate sham illumination (absence of Rose Bengal injection i.p.) has no evidence of infarct. If an infarct is present, the laser illumination intensity or duration is excessive. Misplaced or loose fibers can be quickly identified based on aberrant lesions, and those animals can be excluded from the study. Importantly, varying the intensity and duration of photothrombotic illumination allows one to scale the photothrombosis-evoked neuronal Ca2+ influx from transient to persistent depolarization (Figure 3, see also23). This suggests that lower laser powers and shorter durations initiate transient occlusions (similar to a transient ischemic attack in humans) while longer and more intense illumination triggers permanent occlusions.
<img alt="Chromatography result graph, showing signal intensity over time with variable conditions." class="xfigimg" src="/files/ftp_upload/69418/69418fig3v2.jpg" data-altupdatedat="1778558422000" data-alt="Figure 3"> 
Figure 3: Dose-response of photo-illumination mediates transient to persistent occlusion and ischemia-evoked depolarization. Representative traces from behaving Thy1-GCaMP6f mice receiving increasing photothrombotic illumination duration and power (red: 6 mW, 8 min; black: 6 mW, 1.5 min; blue: 5 mW, 1.5 min; magenta: 4 mW, 1.5 min), revealing respective increases in neuronal Ca2+ influx (ΔF/F). <a href="https://www.jove.com/files/ftp_upload/69418/69418fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are several important controls to consider while setting up an experiment. First, a vehicle 'sham' control is common to photothrombosis experiments, where sterile saline is injected at an equivalent volume per weight, but in the absence of 0.5% Rose Bengal. Here, surgical and recovery practices are identical, and the mouse experiences identical illumination conditions in the absence of Rose Bengal. While the peak of the excitation spectrum of Rose Bengal in an aqueous solution sits between 540-570 nm, it is also weakly excited by 465 nm light (&lt;10% of absorption maximum), the Ca2+-sensitive wavelength used for GCaMP6f photometry. Importantly, the power output of the light source used for photometry (&lt;30 µW, 465 nm) is orders of magnitude lower than that used for photothrombosis (&gt;4 mW, 561 nm). A second control involves injecting 50 mg/kg Rose Bengal i.p. and recording photometry for an equivalent session without 561 nm photo-illumination. This confirms that sustained 465 nm (&lt;30 µW) excitation in the presence of Rose Bengal, without photo-illumination with 561 nm, is insufficient to initiate stroke (Figure 4). In each case, a successful negative control will be absent of significant changes in GCaMP6f photometry, indicative of TD (Figure 3), as well as no evidence of a lesion (TTC stain).
<img alt="Photodynamic therapy graph; Rose Bengal vs. vehicle; illumination effect; experimental results." class="xfigimg" src="/files/ftp_upload/69418/69418fig4v2.jpg" data-altupdatedat="1778558422000" data-alt="Figure 4"> 
Figure 4: Controls for combined photothrombosis and photometry in behaving mice. Representative GCaMP6f traces for a 40 min recording of behaving Thy1GCaMP6f mice experiencing 'Sham' (saline vehicle i.p. (0.01 mL/g) with 561 nm illumination, 8 min, 6 mW) and 'Rose Bengal only' (50 mg/kg i.p. without 561 nm illumination) control conditions and TTC stained coronal sections (48 h post-stroke) demonstrate the absence of terminal depolarization and infarct in both conditions. <a href="https://www.jove.com/files/ftp_upload/69418/69418fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This approach allows induction of stroke in behaving mice, thus behavioral analysis can be performed simultaneously with the TD, and was recently used in combination with behavioral state analysis and overhead center-of-mass locomotion tracking23 (analysis code is freely available here: https://doi.org/10.5281/zenodo.8303055). During hippocampal stroke, mice begin to ambulate around the arena for up to several minutes following the maximum derivative of the GCaMP6f trace (TD, Figure 2A). This hippocampal SD-induced ambulation has been observed in the context of stroke23, following intrahippocampal injection of opioid peptide analogues29, as well as in a model of temporal lobe epilepsy30.

Using an all-optical approach, ischemic stroke is induced while simultaneously recording neuronal Ca2+ activity using a fluorescent biosensor in the emerging stroke core of an awake, behaving mouse.

Stroke is a leading cause of death and disability across the globe. During ischemic stroke, an obstructed cerebral blood vessel causes focal metabolic ...

We're interested in mechanisms of memory impairment, and resilience following stroke. Most preclinical models that investigate stroke related neurophysiology require anesthetic or head fixation, which makes behavioral assessment limited. This approach addresses those issues.To begin, use a digital scale to weigh the mouse. Transfer the anesthetized mouse to the nose cone in the stereotaxic frame to maintain anesthesia. Using an electric razor, remove the hair from the top of the head, starting near the nape and extending towards the eyes.Next, secure mouse in ear bars. Then use a cotton tipped applicator to apply lubricating eye ointment to both eyes. Then clean the dorsal surface of the mouse's head with three serial applications of iodine, followed by 70%ethanol.Now, pinch the toe and observe for any withdrawal reflex and check for blinking to verify sufficient anesthetic depth. Afterward, inject a buprenorphine solution intraperitoneally at a dose of 0.01 milligrams per kilogram. Then make an approximately three centimeter anterior to posterior incision along the scalp to expose the skull.Using a cotton tipped applicator, apply 5%hydrogen peroxide to the dorsal skull to remove the periosteum. Then adjust the stereotaxic frame and ear bars to level the skull in both the dorsal ventral and medial lateral planes. Now, position the tip of the stereotaxic drill above bregma and adjust its anterior, posterior, and medial lateral location to the desired hippocampal coordinates.Lower the stereotaxic drill to the skull and drill a small burr hole using short pulses. Apply ice cold saline intermittently to prevent heating of the skull and brain surface. Repeat until the burr hole is through the skull and the surface of the brain is exposed.Lower the optical fiber until it just contacts the surface of the brain without penetration. Slowly lower the optical fiber just above the CA1 region. Using a precision applicator, apply a quick air dry dental cement to cover the entire exposed skull and the lower one third of the fiber optic cannula to secure it in place.Next, apply UV cured cement to support stability of the implanted fiber. Suture the skin surrounding the implanted fiber optic cannula and hardened dental cement. After turning off the isoflurane vaporizer and loosening the nose piece, transfer the mouse to a heated recovery cage.Turn on the 561-nanometer fiber coupled laser and toggle to TTL mode. Using a digital handheld optical power meter, adjust the 561-nanometer laser power to six milliwatts. Now, turn on the fiber photometry console, the light emitting diode drivers and the detectors.Open the fiber photometry acquisition software. Click on the device selection tab and open NC500 console. Under the add channels tab, add lock-in modulation channels for 465 nanometer and 405 nanometer LEDs using preset frequencies.Match the output voltage of the 405 nanometer LED to approximate the voltage of the 465 nanometer LED. Add a digital input output channel to configure an eight-minute-long square pulse for the TTL input to the 561-nanometer laser. With a 10-minute starting delay.Add a camera channel to set the overhead behavioral camera to be initiated simultaneous with photometry at 20 hertz. Adjust the overhead camera to ensure the entire behavioral arena is clearly visible in the frame. Weigh the mouse using a digital scale.Using a lint-free wipe sprayed with 70%ethanol, scruff the mouse and clean the fiber optic cannula by wiping away any debris. For stroke mice, inject 0.5%rose bengal solution via intraperitoneal injection. While holding the mouse by the scruff, connect the fiber optic patch cord to the implanted feral by twisting the sleeve gently while applying even pressure.Place the mouse into the recording arena and press record to initiate synchronized data collection. After 40 minutes, stop the recording in the photometry acquisition software. Turn off the 561-nanometer laser.Pick up the mouse and carefully detach the fiber optic patch cord by securing the head, then holding the sleeve and using a gentle twisting motion. A sustained increase in neuronal calcium concentration was observed during terminal depolarization in mice injected with rose bengal and subjected to photothrombotic illumination with subsequent infarct formation Under the fiber optic tip. In sham mice injected with saline instead of rose bengal, there was no increase in neuronal calcium and no infarct was detected by TTC staining.The absence of a biosensor, such as in cases of incorrect genotype or misplaced fiber, was indicated by little to no photobleaching at the start of the photometry recording. In contrast, recordings with successful biosensor expression showed an initial downward slope consistent with photobleaching. An optimal hippocampal recording displayed a stable baseline with spontaneous signal transient.The absence of spontaneous transience in the photometry trace suggested biosensor expression failure or incorrect fiber placement. Mice subjected to lower power and shorter photothrombotic illumination showed only transient calcium influx, while those exposed to longer and higher intensity illumination displayed a sustained increase. Both control mice injected with vehicle and exposed to illumination and mice injected with rose bengal but not illuminated, showed no appreciable changes in GCaMP6f signal and no infarct.We identified that depolarization spread through the hippocampus during acute stroke and impair spatial navigation as well as memory. We can now identify the relationship between neurophysiological changes in the hyperacute phase of stroke and how they relate to behavior and recovery. This protocol introduces the ability to simultaneously monitor brain activity dynamics and stroke symptomology in freely behaving mice.

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JoVE Journal

Neuroscience

This video demonstrates the procedure to generate photothrombotic stroke in a mouse model by laser illumination of an intact skull following administration of photosensitive dye.

Modeling Photothrombotic Stroke in Mouse Model: A Laser Illumination Technique Through Mouse Skull Following Photosensitive Dye Administration to Induce Photothrombosis

Source: Talley Watts, L., et al. Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke. J. Vis. Exp. (2015)This video demonstrates a technique to induce blood clot formation in a mouse brain. An anesthetized mouse with a cranial window and a thinned skull undergoes photothrombosis through the activation of a photosensitive dye using high-intensity light. This process generates reactive oxygen species, which damage the vessel walls and trigger clot...

 Inducing Blood Clot Formation in the Mouse Brain Using a Photosensitive Dye

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo.  The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke

This protocol provides an integrated method that includes viral delivery, optrode implantation, photothrombotic induction, simultaneous recording of astrocyte and neuronal activity, and optogenetic stimulation of astrocytes. This integrated method aims to provide concurrent investigation of the astrocyte-neuron relationship across the acute-to-chronic stages of ischemic stroke.

An Integrated Method for Photothrombotic Stroke Modeling and In Vivo Optrode Recording of Neuronal and Astrocytic Activity in Behaving Mice

Learn to induce ischemic stroke in behaving mice while monitoring neuronal calcium dynamics using fiber photometry. This method enables simultaneous study of neurophysiological changes and behavioral outcomes.

Simultaneous Photothrombosis and Fiber Photometry to Induce and Monitor Ischemic Stroke in Behaving Mice

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