Name: Microvascular Embolism Mouse Model for In Vivo Two-photon Microscopy Using Fluorescent Polystyrene Microspheres (Video) | JoVE Journal
Uploaded: 2025-11-21T12:40:00.000+00:00
Duration: 8 min 29 s
Description: Learn to create a microvascular embolism mouse model using fluorescent polystyrene microspheres for in vivo two-photon microscopy. This method enables real-time tracking of capillary plugging and investigation of cerebral blood flow dynamics.

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5 minutes

The authors would like to express their sincere gratitude to Dr. Iwan Dobbe for developing the Articulus software, which was used for the in situ 3D image reconstructions. We also thank Prof. Dr. Nikolaus&#160;Plesnila for kindly providing TIE2-GFP mice (Jackson Laboratory, Stock No. 003658) used for in-house breeding.
This study was funded by Amsterdam Neuroscience (813294 to I.M.), Amsterdam Cardiovascular Sciences (Equipment call 2021 and 2023 to I.M.) and the Dutch Heart Foundation (03-006-2021-T019 to I.M.).&#160;
This research is also funded by the Dutch Heart Foundation and The Brain Foundation Netherlands and is conducted in collaboration with and supported by the Dutch CardioVascular Alliance, 01-002-2022-0126 CONTRAST 2.0. In addition, this work was funded in part through unrestricted funding by Stryker, Medtronic and Penumbra. The funding sources were not involved in study design, monitoring, data collection, statistical analyses, interpretation of results, or manuscript writing.

The authors declare no conflicts of interest.

To model microvascular embolisms, a protocol for injecting microspheres into the cerebral circulation of mice was described that allows the study of micro-embolism using in vivo two-photon microscopy. This method optimized microsphere entrapment in the brain, with multiple microspheres trapped within the first 500 &#181;m of the ipsilateral cortex, enabling effective two-photon imaging.
A high microsphere count in the cortical area of the brain is crucial for in vivo two-photon imaging. The loss of microspheres was minimized during the preparation of the microsphere mixture by coating the pipette tips with 5% BSA or 0.1% Tween20, reducing microsphere adhesion (see Supplementary File 1). While the exact number of adhered microspheres could not be quantified, mean intensity measurements showed the effect of the coatings. Additionally, Tween20 appeared to prevent clustering of microspheres in the catheter, resulting in a visually higher number of singular microspheres flowing through the catheter, although this is not quantified. This reduces the risk of large vessel occlusions and the potential formation of large ischemic and hypoxic regions, which is undesirable given the objective to model microvascular embolizations. Moreover, mice exhibited less weight loss and recovered to pre-surgical weights more rapidly when injected with a microsphere mixture containing low concentrations of Tween20. Tween20 is a commonly used dispersant45,46,47, therefore, the low concentrations used in this study are not expected to cause any toxicity or adverse effects on BBB integrity. In addition, our data shows that BBB leakage occurs only near (or after passing of) the microsphere lodging and not systematically, while not causing a significant increase in total leukocyte coverage (see Supplementary File 2), supporting the use of 0.1% Tween20. Overall, the addition of Tween20 contributes to reduced clumping of microspheres and an increase in microsphere delivery to the cerebral circulation, confirmed by microsphere counts in brain slices, and results in sufficient microspheres visible for two-photon microscopy. Although the use of Tween20 increases the number of microspheres lodged in the brain, variability in microsphere injection remains. Several factors may contribute to this variation. First, thrombus formation within the catheter or vasculature can cause microspheres to adhere to the clot. Second, the presence of air bubbles may interfere with smooth delivery. Finally, incomplete restoration of blood flow in the CCA or ICA can alter hemodynamics, potentially promoting microsphere adhesion to the vessel wall or clustering within the lumen. Unfortunately, these deviations cannot be detected during the injection procedure.
Polystyrene microspheres are denser than water, leading to uneven distribution once the microsphere mixture is loaded into the catheter. Most microspheres settle near the catheter wall, where fluid friction slows their movement relative to the faster central flow. This eventually results in an accumulation of microspheres within the final 20&#8239;&#181;L of the catheter. Injecting this concentrated portion should be avoided, as it may cause large emboli and ischemic events. To prevent this, the microsphere mixture volume should be prepared at twice the intended injection volume.
The microvascular embolism surgery demands substantial training and precision. One of the most technically challenging steps involves placing a suture around the PPA, the OA, and side branches of the ICA to ensure accurate delivery of the microsphere mixture into the circle of Willis and the MCA territory. This step is complicated by the close proximity of the vagus nerve near the CCA, the small diameter of the vessels and limited surgical space, making it difficult to position the sutures without causing nerve damage or vascular injury.
While a vessel clip may be considered as an alternative to temporarily ligating the PPA using a suture, it introduces additional risks. Clips can unintentionally detach, leading to vessel reopening. Moreover, using a second clip at the PPA alongside the existing clip on the ICA, increases the likelihood of detachment of the ICA clip due to limited space. If this occurs, it will cause a significant hemorrhage and, without immediate intervention, can result in early termination of the experiment. Therefore, closure of the PPA using a vessel clip is not recommended.
The current protocol employs a catheter, rather than a syringe33, for microsphere injection. This approach offers better control over injection speed and volume, and it enables injection of the microspheres more remotely (e.g., while inside an imaging modality such as a microscope or MRI scanner).
Choosing the ECA over the CCA as the entrance point enables the CCA to stay fully open during the injection procedure, which helps maintain a more physiological cerebral perfusion throughout the surgical procedure. In addition, injecting the microspheres via the ECA instead of the CCA has other advantages48: i) reduced total operation time during reperfusion; ii) decreased risk of bleeding during injection and after surgery completion; iii) with the CCA-entrance method, an unstable stenosis and thrombosis is formed in the CCA after removing the needle49. This will cause disturbed blood flow, as well as the risk of microthrombi formation, which can end up in the brain. Additionally, entering via the CCA would involve a large risk of permanently closing the CCA, which can result in sub-optimal cerebral reperfusion during the survival period after surgery, worsened by the high variation in the circle of Willis in mice50,51
Although fully optimized, the protocol has some limitations. First, the observation that microspheres travel more slowly along the catheter walls and are not evenly distributed during injection suggests that fewer microspheres are delivered into the ICA than expected. Despite the use of Tween-20 to reduce adherence, some microspheres will still stick to the pipette tips, Eppendorf tube, and catheter. Consequently, the actual number of microspheres injected is likely lower than the calculated 7.2 x 104&#160;particles per mouse. Nonetheless, sufficient microspheres reach the cerebral circulation to allow for imaging via two-photon microscopy. The injection protocol and mixture composition are optimized for 10&#8239;&#181;m polystyrene microspheres. If the materials used (tips, tubes, catheters) or the size of the microspheres are altered, both the solution and injection procedure should be re-evaluated accordingly. Second, studies investigating the best method for large vessel occlusion via intra-luminal occlusion of the MCA have shown that entrance of the vascular system via permanent ligation of the ECA results in ischemia of the masticatory and swallowing muscles52. This can result in decreased food intake of mice and, therefore, excessive weight loss, impaired motor function, and increased neurological deficit. However, this statement has been contradicted by others48. In this study, the weight monitoring showed an expected drop after surgery but a fast recovery to pre-surgical weights within several days, indicating that the mice do recover and eat and drink normally after permanent ECA ligation. Although the weight of the animals was monitored, this is only an indirect proxy of the animals wellbeing and does not fully rule out functional impairments like local masticatory or motor deficits. No direct assessments on functional recovery were performed, which is a limitation of this study. Another study compared the entrance of the vasculature system via the CCA or ECA and showed that entry via the CCA can result in impaired reperfusion of the cerebral circulation, concluding that the ECA entry method is preferred53. Third, proper and secure catheter placement without introducing air bubbles, blood clots, or leakage, and without obstructing flow in the CCA and ICA requires significant practice. Last, in contrast to other micro-occlusion methods like photothrombosis using Rose-Bengal injection22, one cannot predict where the microspheres will eventually lodge in the circulation.
The present study deliberately does not include negative (sham) or positive (photothrombosis) control groups, as these would not provide additional value for the primary objective. The success of this model is defined by the number of microspheres lodged within the cerebral microcirculation, particularly in the cranial window area, to enable high-resolution in vivo microscopy. A sham procedure would, by definition, yield no microspheres in the target area and thus offer no relevant comparison for the in vivo imaging endpoint. Similarly, inclusion of a photothrombosis model would address a fundamentally different pathophysiological mechanism and is therefore beyond the scope of this work, which focuses on characterizing the microvascular embolism model rather than comparing methodologies. Broader histological analyses of embolisms and their potential effect on the BBB or local neuronal tissue were not pursued, as the study was designed to prioritize in vivo 3D visualization over tissue-level validation.
The detailed surgical protocol for both the cranial window preparation and microvascular embolism model supports reliable and reproducible investigation of microvascular changes associated with microvascular embolism and the occurrence of silent brain infarcts throughout the ipsilateral hemisphere. The optimized preparation and injection of the microsphere mixture further enhance the consistency and validity of this experimental approach.

An ischemic stroke due to embolism of a large cerebral artery is an acute and often devastating event1. Emboli may also occur in more distal vessels. As a result, silent brain infarcts (SBIs) may develop; as the name suggests, these remain asymptomatic, and are typically discovered incidentally during imaging for unrelated reasons2,3. SBIs are common and rank among the most frequently observed cerebrovascular conditions in autopsies of elderly individuals4,5. Epidemiological studies have shown a 20% prevalence in individuals without a history of stroke. SBIs contribute to cognitive dysfunction and dementia, increase overall mortality, and are a risk factor for future stroke6,7,8. The infarcts are typically only a few millimeters in diameter and are associated with occlusion of penetrating arteries with diameters of roughly 100 &#181;m. These penetrators give rise to an extensive arteriolar and capillary bed. A small microthrombus passing through the penetrating arteries and lodging in the downstream microcirculation will likely remain undetectable on angiography9. Yet, many microthrombi may be released in atrial fibrillation, atherosclerotic plaques, and from other sources10,11,12,13,14. Besides microthrombi, other microparticles (MPs), including fat particles, cellular aggregates, and even microplastics, may cause microvascular embolisms. Several studies show the presence of plastic MP in the blood and the brain that are larger than the capillary diameter of 5 &#181;m15,16.
Microvascular occlusions remain relatively understudied compared with acute stroke and SBI. Little is known about the event rate, long-term consequences, and possible clearing mechanisms. Yet, interest is rapidly increasing after the successful clinical introduction of mechanical thrombectomy in stroke, where microvascular embolisms may cause impaired microvascular reperfusion after apparently successful recanalization of the large culprit artery17. In addition, there is a surge in research on microplastics, which may partly have their effect through microvascular embolism18,19,20.
Microvascular occlusions in the mouse cortex can be studied by, among others, multi-photon microscopy, using a cranial window. The most commonly used models to study cerebral microvascular occlusions are the photothrombotic and the microsphere model. The photothrombotic model employs photosensitive dyes to induce embolisms in selected vessels (typically non-capillary vessels) via photoactivation. However, this model does not allow the study of embolisms over a longer period due to the activation of the fibrinolytic system, which dissolves the embolism21,22. Moreover, activation of the light-sensitive dye produces singlet oxygen species, causing endothelial and blood-brain barrier (BBB) damage23. For studies on endothelial and BBB function during cerebral embolisms, and the long-term effects on cerebral circulation and brain function, the microsphere model is therefore preferred. The microsphere injection model uses microspheres injected into the cerebral circulation to occlude the cerebral microvasculature9,24,25,26,27,28,29,30. The possible disadvantage of this model lies in the fact that it is not possible to predetermine the location of embolisms, in contrast to the photothrombotic method31,32. The advantage is that microspheres of well-defined diameters can be injected, thereby targeting (micro-) vessels of specific diameters33,34,35.
Over the years, multiple approaches for the injection of microspheres into the cerebral circulation of rodents have been developed. These protocols find consensus in accessing the carotid triangle, consisting of the common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) for intra-arterial microsphere injection. The method for intra-arterial microsphere injection varies between protocols in several ways. Firstly, studies used either a syringe9,26,27,29,33,34 or a catheter25,28,30,36. Secondly, there is a considerable variation in the number and size of injected microspheres. Microsphere diameters typically range between 10 and 50 &#181;m, with the choice depending on the target vessel size. As a rule of thumb, the larger the microspheres, the lower the number one should inject, as there are fewer arterioles than capillaries25,26,30.
Achieving consistent intra-arterial microsphere injection remains challenging. In practice, large numbers of microspheres are injected, yet only a fraction reaches the cerebral circulation. This is especially problematic for studying the effects of lodged microspheres by in vivo two-photon microscopy imaging, as the success of this technique depends on the presence of microspheres in the outer layers of the mouse cortex within the view through the prepared cranial window. Choosing appropriate fluorescent dyes for microspheres is critical, as combining them with transgenic mouse models and intravascular staining can be challenging due to the intense fluorescence of the microspheres and overlapping emission spectra.
Microvascular occlusions are studied in rodent models, and synergistic effects of multiple microspheres have previously been demonstrated in ex vivo rodent brains9, as well as apparent extravasation of such particles, a process called angiophagy33,35,37,38. To enable their in vivo investigation, methods were developed for multi-photon microscopy in mice with cranial windows. The purpose of the current study is to develop and report a detailed protocol for intra-arterial injection of microspheres in mice that optimizes the study of their effects on the brain tissue and their fate. The main advantage of this protocol is its improved delivery efficiency, minimizing microsphere loss and increasing the likelihood of their presence in the middle cerebral artery (MCA) territory within the cranial window imaging area. The optimized microsphere injection protocol is evaluated using in vivo two-photon microscopy and post mortem in situ 3D imaging with a dedicated imaging cryo-microtome39.

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2.3.3.)</td></tr><tr><td>Micro vascular clip</td><td>Braun-Aesculap</td><td>FD562R</td><td>Vessel clip (Step 2.6.1.)</td></tr><tr><td>Micro-catheter (ID 0.23114 mm OD 0.27432 mm)</td><td>Microlumen</td><td></td><td>Micro-catheter (Step 2.1.1.)</td></tr><tr><td>Mouse Heating Pad and Rectal Probe</td><td>Stoelting</td><td>53800R</td><td>Heating pad (Step 1.1.2. &amp;amp; 2.2.2.)</td></tr><tr><td>NG2-DsRed mice</td><td>Jackson Laboratory, Bar Harbor, USA, Stock No. 008241</td><td></td><td></td></tr><tr><td>Polyethylene glycol sorbitan monolaurate</td><td>Sigma-Aldrich</td><td>9005-64-5</td><td>Tween20 (Step 2.5.2.)</td></tr><tr><td>Polystyrene FluorSpheres Blue (365/415)</td><td>Invitrogen</td><td>F8829</td><td>Blue microspheres (Step 2.5.1.)</td></tr><tr><td>Polystyrene FluorSpheres red (580/605)</td><td>Invitrogen</td><td>F8834</td><td>Red microsphere (Step 2.5.1.)</td></tr><tr><td>Recovery cage</td><td>-</td><td>-</td><td>Recovery cage (Steps 1.6.2. &amp;amp; 2.8.2.)</td></tr><tr><td>Reverse cutting 3/8 curved needle (12mm) with suture</td><td>Bbraun</td><td>0762067</td><td>Suture (Step 2.7.5)</td></tr><tr><td>Ropivaca&amp;iuml;ne (2 mg/ml )</td><td>-</td><td>-</td><td>Medication&amp;nbsp; (Step 1.1.5.)</td></tr><tr><td>small 3D Fluorescent Imaging Cryomicrotome&amp;nbsp;&amp;nbsp;</td><td>Custom made</td><td></td><td>Small-3D-FICS (Step 4.2.2)</td></tr><tr><td>Spongostan</td><td>Ethicon</td><td>MS0005</td><td>Spongostan (Step 1.4.6.)</td></tr><tr><td>Surgical microscope</td><td>Euromex Microscopen B.V.</td><td>NZ.1903-B</td><td>Microscope for surgery (Step 1.2. 2.1.1.)</td></tr><tr><td>Surgical scissors</td><td>Braun-Aesculap</td><td>BC303R</td><td>Surgical scissors (Step 1.2.2. &amp;amp; 2.3.2.)</td></tr><tr><td>Syringe pump (for 1 ml syringes)</td><td>Havard apparatus</td><td>70-2208</td><td>1 ml syringe pump (Step 2.5.6.)</td></tr><tr><td>Thread, 100m,&amp;nbsp; 4/0 1.5&amp;nbsp;&amp;nbsp;</td><td>Bbraun</td><td>F1134035</td><td>4/0.15 threads (Step 2.4.3.)</td></tr><tr><td>TIE2-GFP mice</td><td>Jackson Laboratory, Bar Harbor, USA, Stock No. 003658</td><td></td><td></td></tr><tr><td>Ultra pure duster</td><td>Thorlabs</td><td>CA6-EU</td><td>Compressed air (Step 1.2.5.)</td></tr><tr><td>Universal Polymer Clear</td><td>Sun Medical</td><td>T058E</td><td>Component of super glue (Step 1.3.1.)</td></tr></tbody></table>

microvascular embolism mouse model for in vivo two-photon microscopy using fluorescent polystyrene microspheres

Microvascular Embolism Mouse Model for In Vivo Two-photon Microscopy Using Fluorescent Polystyrene Microspheres

Microvascular occlusions in the brain are relatively common but remain largely understudied. The event rate, long-term consequences, and potential clearing mechanisms are largely unknown. Current models used to study these events, such as photothrombosis and micro-emboli injection, each have their own advantages and limitations. This study developed a detailed protocol that combines the preparation of a chronic cranial window with the induction of cerebral microvascular embolisms via intra-arterial injection of microspheres in mice. This setup allows for long-term in vivo imaging of the micro-vasculature and micro-occlusions using two-photon microscopy. Microspheres are delivered through a catheter placed in the external carotid artery (ECA), which is permanently ligated. Importantly, the common carotid artery (CCA) and internal carotid artery (ICA) remain intact throughout and after the procedure, thereby minimizing disturbance to cerebral blood flow. To facilitate immediate in vivo imaging and prevent microsphere clustering or adhesion to pipette tips and the catheter, microspheres are suspended in a mixture of FITC-Dextran and 0.1% Tween 20. The injection technique was validated using post mortem in situ 3D imaging to determine the microspheres&#39; distribution. This study further demonstrates its utility with an in vivo two-photon microscopy example. This approach provides a consistent and robust method for inducing and studying microvascular embolisms, enabling investigation of their impact and clearance dynamics using high-resolution in vivo imaging.

Post mortem in situ imaging
The success of the microvascular embolism surgery was confirmed by full-body and full-brain post mortem in situ imaging 4 days post-microvascular embolism surgery (Figure 3A). The sagittal slice of a mouse shows lodging of microspheres in the brain. Figure 3B,C show an example of an extracted mouse brain, 1 day post-microvascular embolism surgery. Microspheres were predominantly lodged in the ipsilateral hemisphere, in the flow territory of the middle and anterior cerebral arteries. Microsphere counts in brain slices were significantly increased when Tween20 was added to the mixture (Figure 3E). Monitoring the weight during the recovery after the micro-embolism surgery shows that mice lost less weight and recovered faster after surgery when injected with a microsphere mixture that contained Tween20 (Figure 3F).
<img alt="Sagittal whole mouse image with microsphere distribution, brain plane views, and tissue sphere analysis." class="xfigimg" src="/files/ftp_upload/68939/68939fig3v2.jpg" data-altupdatedat="1783396841000" data-alt="Figure 3"> 
Figure 3: Distribution of microspheres in the mouse after microvascular embolism surgery. In situ localization of 10 µm microspheres in the mouse visualized using Articulus software41. (A) Sagittal plane example of a whole mouse imaged with the large-3D-FICS after microvascular embolism surgery. (B-D) Representative axial, coronal, and sagittal images of a mouse brain, imaged using the small-3D-FICS. (E) Average number of detected microspheres per 50 µm coronal brain sections at approximately bregma ± 0.5 mm for the Control and Tween20 conditions. Bars represent mean ± standard deviation. Individual measurements are shown as dots. Statistical comparison was performed using an unpaired t-test; the p-value is indicated in the graph. Outliers were identified and removed using the interquartile range (IQR) method (n = 1 for both groups), resulting in a final group size of n = 3 for controls and n = 14 for Tween20. (F) Normalized weight changes over time following microvascular embolism surgery at timepoint 0. Weights were normalized to each animal's baseline at surgery. Lines show mean ± SD per group (Control, n = 4; Tween20, n = 5) with Control in blue and Tween20 in dark orange. Statistical significance between groups at each time point was assessed using unpaired two-tailed t-tests. (* = p &lt; 0.05, ** = p &lt; 0.01) <a href="https://www.jove.com/files/ftp_upload/68939/68939fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Microsphere injection and in vivo two-photon microscopy
During microsphere injection, catheters coated with 5% BSA showed many microspheres adhered to the catheter surface, and flowing microspheres were often clustered, which may potentially cause larger occlusions (see Supplementary Video 1). In addition, adding 5% BSA in the microsphere mixture to prevent adhesion and clustering is not desirable due to a potential immune response upon injection43,44. To circumvent this, a 0.1% Tween20 solution was used in the microsphere mixture, resulting in a visibly higher number of microspheres flowing through the catheter and in the desired number (&gt; 20) of microspheres in the imaging window (Figure 4 and Supplementary Video 2). Figure 4A,B shows a fluorescence microscopy overview image of the brain cortex 0.5 h after the microvascular embolism surgery. Each white dot represents a microsphere that occludes a capillary in the cerebral microvasculature. Figure 4C,D displays the two-photon z-stack images 2D projection as an example. Microspheres are causing cerebral microvascular embolisms with impaired perfusion, indicated by the absence of intraluminal dye downstream of the microsphere (Figure 4E and Supplementary Video 3). Additionally, these embolisms cause disruption of the BBB with extravasation of the FITC-Dextran dye (Figure 4F,G).
These results are in accordance with the low number of microspheres adhered to the catheter and minimal microsphere clustering, which was observed in the catheter while injecting the microsphere-Tween mixture. However, a large number of microspheres did cluster together in the final 20 µL of the microsphere mixture in the catheter, which should not be injected to prevent the possible occurrence of a large infarction (see Supplementary Video 4).
<img alt="Fluorescent microscopy showing cerebral microvascular embolization with green vessels, pink microspheres." class="xfigimg" src="/files/ftp_upload/68939/68939fig4.jpg" data-altupdatedat="1783396841000" data-alt="Figure 4"> 
Figure 4: Two-photon in vivo microscopy of lodged microspheres. Examples of in vivo fluorescent and two-photon images of lodged microspheres in the mouse cortex. (A) Fluorescent overview through the cranial window of the lodges microspheres. (B) Fluorescent image of the selected ROI. (C,D) Two-photon microscopy images of entrapped microspheres in the cerebral microcirculation with selected ROI for detailed imaging. Z-stack images are depicted in 2D by calculating the standard deviation of pixel intensities along the Z-stack. (E-G) Representative in vivo two-photon microscopy images showing three cerebral microvascular embolisms. Red arrows highlight areas of FITC-Dextran extravasation, indicating BBB disruption and dye leakage. <a href="https://www.jove.com/files/ftp_upload/68939/68939fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: Two-photon microscope system specifications. <a href="https://www.jove.com/files/ftp_upload/68939/JoVE68939_Supplementary%20table%201.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Table 2: Two-photon microscope settings for FITC-dextran and microsphere imaging. <a href="https://www.jove.com/files/ftp_upload/68939/JoVE68939_Supplementary%20table%202.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Table 3: Small 3D Fluorescent imaging Cryomicrotome system settings for mouse brain imaging with blue microspheres. <a href="https://www.jove.com/files/ftp_upload/68939/JoVE68939_Supplementary%20table%203.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Table 4: Small 3D Fluorescent imaging Cryomicrotome system specifications for mouse brain imaging with blue microspheres. <a href="https://www.jove.com/files/ftp_upload/68939/JoVE68939_Supplementary%20table%204.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Table 5: Large 3D Fluorescent imaging Cryomicrotome system settings for whole mouse imaging with red microspheres. <a href="https://www.jove.com/files/ftp_upload/68939/JoVE68939_Supplementary%20table%205.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Table 6: Large 3D Fluorescent imaging Cryomicrotome system specifications for whole mouse imaging with red microspheres. <a href="https://www.jove.com/files/ftp_upload/68939/JoVE68939_Supplementary%20table%206.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Video 1: 5% BSA-coated catheter with microsphere adhesion to the catheter wall and microsphere clustering in the 25 mg/mL of FITC-Dextran microsphere mixture during injection. <a href="https://www.jove.com/files/ftp_upload/68939/JoVE68939_Supplementary%20video%201.mp4" target="_blank">Please click here to download this video.</a>
Supplementary Video 2: 0.1% Tween20-coated catheter with some microsphere adhesion to the catheter wall but without microsphere clustering in the 25 mg/mL FITC-Dextran with 0.1% Tween20 mixture with microspheres during injection. <a href="https://www.jove.com/files/ftp_upload/68939/JoVE68939_Supplementary%20video%202.mp4" target="_blank">Please click here to download this video.</a>
Supplementary Video 3: 3D segmentation of microvascular occlusions by microspheres. <a href="https://www.jove.com/files/ftp_upload/68939/JoVE68939_Supplementary%20video%203.mp4" target="_blank">Please click here to download this video.</a>
Supplementary Video 4: Final volume of the microsphere injection with high microsphere concentration in 25 mg/ml FITC-Dextran with 0.1% Tween20 mixture. <a href="https://www.jove.com/files/ftp_upload/68939/JoVE68939_Supplementary%20video%204.mp4" target="_blank">Please click here to download this video.</a>
Supplementary File 1: Microsphere adherence to pipette tips validation. <a href="https://www.jove.com/files/ftp_upload/68939/JoVE68939_Supplementary%20file%201.pdf" target="_blank">Please click here to download this file.</a>
Supplementary File 2: Immunohistochemical assessment of CD45 coverage at 1, 7, and 28 days post-microembolism surgery comparing the ipsilateral and contralateral hemispheres. <a href="https://www.jove.com/files/ftp_upload/68939/JoVE68939_Supplementary%20file%202.pdf" target="_blank">Please click here to download this file.</a>

This protocol contains a thorough description of a step-by-step approach for a chronic cranial window and microvascular embolism mouse model optimized for in vivo two-photon microscopy imaging using fluorescent polystyrene microspheres.

Microvascular occlusions in the brain are relatively common but remain largely understudied. The event rate, long-term consequences, and potential ...

Using this microsphere injection method, we aim to unravel the short and long-term consequences of capillary plugging to investigate potential mechanisms of clearing of embolus in cerebral capillary network. It allows for real-time in vivo imaging and tracking of fluorescent particles in the cortical capillary network. The microspheres and the local consequences can be followed for weeks after injection.Our studies help to understand local tissue damage caused by microvascular occlusions. These results may hopefully lead to new treatment strategies. We can now study day by day and for weeks how capillary plugging alters vessels, activates cells, changes blood flow, and damages the blood-brain barrier.To begin, pinch the toe of an anesthetized mouse to verify the depth of anesthesia. Make a midline incision of approximately 0.5 centimeters over the trachea below the mandible using surgical scissors and dissecting forceps. Dissect the connective tissue superficial to the cervical fascia using micro-suture-tying forceps to expose the underlying sternal hyoid muscles.Now, separate the left and right sternal hyoid muscles by gently tearing the connective tissue between them. Retract the right omohyoid muscle caudal-laterally with a tissue hook. Ensure that the right carotid triangle, including the common carotid artery, internal carotid artery, and external carotid artery, are clearly visible.Next, remove any remaining fascia and adipose tissue surrounding the common carotid artery. Carefully separate the common carotid artery from the vagus nerve. Place two 4/0 1.5 threads around the common carotid artery and tie them loosely, ensuring the knots do not impede blood flow.Remove fascia and adipose tissue from the internal carotid artery and posterior parietal artery. Then temporarily ligate the posterior parietal artery and all internal carotid side branches, including the occipital artery, by tying a knot with a 4/0 1.5 thread. Remove fascia and adipose tissue from the external carotid artery and the temporal artery.Place two loose knots around the external carotid artery and temporal artery using 4/0 size 1.5 threads. Position the most distal thread as far away from the Y-shaped bifurcation as possible, and use it to permanently ligate the external carotid artery and temporal artery. Leave the proximal knot untied.Homogenize and sonicate the 10 micrometer microspheres to obtain a uniform suspension of individual particles. Add 20 microliters of homogenized microspheres to 140 microliters of FITC-Dextran to obtain a final volume of 160 microliters of mixture containing 1.44 times 10 to the power of five microspheres. Briefly retract the syringe connected to the catheter to create an airlock.Then immerse the catheter tip into the prepared FITC-Dextran/Tween20/microsphere mixture and slowly pull back the syringe until the mixture is in the catheter. Ensure no air bubbles are present in the catheter. Place the syringe with the loaded catheter in the syringe pump.Set the pump to 10 microliters per minute and run until a small drop of microsphere mixture appears at the catheter tip. Now, place a vessel clip on the internal carotid artery and tighten one of the previously prepared proximal knots around the common carotid artery. Using micro-scissors, make a small diagonal incision in the external carotid artery approximately 0.5 millimeters distal to the ligation thread, ensuring the incision size is slightly smaller than the catheter diameter.Absorb any blood with a sterile cloth. Briefly start the syringe pump to form a drop of microsphere mixture at the catheter tip, confirming that no air remains in the catheter tip. Open the external carotid artery incision and insert the catheter using fine micro-forceps.Then secure the catheter in the external carotid artery by tightening the proximal suture around the external carotid artery. Gradually pump at 10 microliters per minute until the artery visibly enlarges and the green FITC dye fills the external carotid artery, confirming successful injection. Next, remove the vessel clip from the internal carotid artery.Then increase the syringe pump flow rate to 20 microliters per minute. Confirm successful microsphere injection by verifying that the internal carotid artery distal to the posterior parietal artery bifurcation is filled with FITC dye. Once injection is successful, remove the thread that ligated the common carotid artery to enable the microsphere mixture to flow into the internal carotid artery.During the injection, the FITC dye can still be observed in the external carotid artery. Switch off the syringe pump once the mixture in the catheter reaches the 80 microliter mark to stop injection. Then ligate the common carotid artery, clip the internal carotid, and gently remove the catheter, while keeping the securing thread in place.Permanently ligate the external carotid artery by tightening the thread that had secured the catheter. Remove the vessel clip and threads around the common carotid artery, internal carotid artery, and posterior parietal artery to restore blood flow. The success of the microvascular embolism surgery was confirmed by full body and full brain post mortem in situ imaging four days after surgery.A sagittal brain slice showed lodging of microspheres in the brain. Extracted mouse brains imaged one day post-surgery showed that microspheres were predominantly lodged in the ipsilateral hemisphere within the flow territory of the middle and anterior cerebral arteries. Microsphere counts in brain slices were significantly increased when Tween20 was added to the microsphere mixture.Mice lost less weight and recovered faster after surgery when injected with a microsphere mixture that contained Tween20. Fluorescence microscopy images of the brain cortex taken 0.5 hours after surgery showed individual white dots, representing microspheres occluding capillaries. Two-photon z-stack projection images showed microspheres causing cerebral microvascular embolisms with impaired perfusion.Embolisms also cause disruption of the blood-brain barrier with visible dye leakage from the vessels.

microvascular-embolism-mouse-model-for-vivo-two-photon-microscopy

Research

JoVE Journal

Neuroscience

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

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

This video demonstrates the intravital fluorescence microscopy technique to study thrombus formation in a mouse model. The mouse is injected with a fluorophore-labeled polysaccharide to monitor blood circulation. Upon illumination with a high-intensity light, the phototoxic induction of an injury in the microvasculature of the ear leads to the formation of a thrombus, which is confirmed by visualizing the obstruction in the blood flow.

Intravital Fluorescence Microscopy to Study Microvascular Thrombus Formation

Source: Jongbloets, B. C., et al. Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy. J. Vis. Exp. (2019) This video demonstrates a two-photon fluorescence lifetime imaging microscopy procedure for visualizing protein kinase A activity in head-fixed, behaving mice during enforced locomotion.

Visualizing Protein Kinase A Activity in a Mouse Using Two-Photon Fluorescence Lifetime Imaging Microscopy 

Source: Nishimura, C. et al. Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation. J. Vis. Exp. (2016). This video demonstrates the in vivo visualization of cortical capillaries in the mouse brain using two-photon microscopy. It outlines the steps for injecting a fluorescent dye into an anesthetized mouse with a thin-skull cortical window, preparing the setup for imaging, and acquiring detailed images of brain...

 Two-Photon Microscopy for In Vivo Imaging of Mouse Brain Microvasculature 

Source: Kim, D., et. al. Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli. J. Vis. Exp. (2016)This video demonstrates visualization of cerebral thrombus in mice using micro-computed tomography (micro-CT) imaging. A cerebral thrombus mouse model is injected with fibrin-targeted gold nanoparticles acting as a contrasting agent. The mouse is placed on the bed of a micro-CT machine, and images are acquired from multiple angles.

Visualization of a Cerebral Thrombus in a Mouse Using Micro-Computed Tomography Imaging

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy.  Intravital microscopy is technically challenging in a moving organ such as the lung.   We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

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

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

We present a detailed protocol for Epon post-embedding correlative light and electron microscopy using a fluorescent protein called mScarlet. This method can maintain the fluorescence and the ultrastructure simultaneously. This technique is amenable to a wide variety of biological applications.

Epon Post Embedding Correlative Light and Electron Microscopy

The protocol describes a method for injecting tumor cells through the percutaneous puncture route into cisterna magna that robustly induces leptomeningeal metastasis in mice, reducing trauma and extracranial tumor burden.

Minimally Invasive Cisterna Magna Injection Model for Leptomeningeal Metastasis Studies in Mice

Learn to create a microvascular embolism mouse model using fluorescent polystyrene microspheres for in vivo two-photon microscopy. This method enables real-time tracking of capillary plugging and investigation of cerebral blood flow dynamics.

Microvascular Embolism Mouse Model for In Vivo Two-photon Microscopy Using Fluorescent Polystyrene Microspheres

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