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Abstract
Ischemic stroke is caused by occlusion of the blood vessels in the brain, where intravenous thrombolytic therapy is the most effective treatment. Urokinase is a commonly used drug for intravenous thrombolytic therapy, while the effect of vessel size has not been thoroughly studied on urokinase. In this work, using the thrombin-combined photothrombosis model and craniotomy-free skull optical clearing window, we studied the recanalization of different cortical vessels after urokinase treatment. The results demonstrated that, compared to small vessels in distal middle cerebral artery (MCA) and large MCA, urokinase has the best therapeutic effect on secondary branches of MCA. This study holds potential to provide references for the clinical applications of urokinase.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Ischemic stroke is a disease that can cause high neurological dysfunction and even death. It is usually caused by the blockage of cerebral blood vessels after thrombosis, and has a high rate of mortality, disability, and recurrence [1–3]. There are many methods for the treatment of ischemic stroke, among which intravenous thrombolysis is the most important clinical thrombolytic method, and the most effective treatment method recognized at present [4,5]. Intravenous thrombolysis involves the recanalization of blood vessels by injecting drugs that dissolve the thrombosis into veins. Urokinase (UK) is one of the commonly used intravenous thrombolytic drug in clinical practice, especially in developing countries, which holds the advantages of low price and long time window [6]. Despite its high thrombolytic efficiency, failure cases still occur occasionally [7,8]. Therefore, it is of great significance to investigate the factors those impact the therapeutic effect of UK to provide the basis for clinical treatment plan.
In the past, researchers have performed a large number of clinical statistical studies and have found that various factors would affect the efficacy of intravenous UK thrombolytic therapy, such as age, gender and basic diseases of the patients, as well as the dosage and method of administration [9–11]. However, the correlation analysis between UK thrombolysis and vascular size has rarely been reported, while it is an important factor that might influence thrombolytic therapy. Researchers studied alteplase, another commonly used thrombolytic drug, in several clinical patients with severe stroke, where they found that for large basilar artery and carotid artery, the recanalization rate was only about 4%, but for the M1 and M2 segments of the middle cerebral artery (MCA), those were relatively higher, the vessel recanalization rate was about 31% [12]. Such results indicates that different size of the blocked vessel might lead to different thrombolysis effect. Therefore, it is valuable to systematically study the influence of vascular size on the therapeutic effect of UK.
To systematically investigate the effect of UK on cerebral vessels with different sizes, a stable animal model that allows targeted ischemic stroke is needed. At present, many animal models have been used in the study of ischemic stroke, such as intraluminal suture model [13], thrombosis-introduced model [14], FeCl3-induced model [15], photothrombotic model [16] and in situ thrombin injection model [17] etc. Among the models above, except for photothrombotic model, other model could hardly achieve targeted embolization of vessels at different locations, especially for small vessels. However, traditional photothrombotic model has limitations in the application of thrombolytic drug evaluation.
The mechanism of the photothrombotic model is to use the photodynamic effect of photosensitizers to cause vascular endothelial cell injury, leading to platelet gathering. However, clinically stroke thrombus usually contains not only platelets, but also a large amount of fibrin, which is the main target of many thrombolytic drugs, including UK. This had made it difficult to study the effects of thrombolytic drugs. Another obstacle is, for targeted photothrombotic modeling on certain vessels, it is necessary to visualize them first. However, the scattering of the turbid skull make it hard to obtain cerebrovascular distribution. To overcome the difficulty, open-skull preparation was often used before modeling in the previous study. However, despite the inconvenience of the surgery operation, skull removal would cause changes in intracranial pressure, and might lead to a series of inflammatory reactions, making the brain in an abnormal environment even before stroke.
Those two obstacles have limited investigations of drug evaluation for targeted ischemic stroke. Fortunately, two novel techniques offer new insights into such studies. Firstly, an optimized photothrombotic model has been proposed lately. By introducing thrombin with the photosensitizer together, the formed thrombus contained both platelets and fibrin, which was proved sensitive to a typical thrombolytic drug, alteplase [18].
Secondly, novel in vivo skull optical clearing technique could make skull transparent by the application of biocompatible reagents to the skull, opening a “optical window” for light manipulation and observation through an intact skull [19–21]. Through the skull optical clearing window, the dynamics of submicron-scale dendritic spines of neurons could be visualized, the light-induced blood-brain barrier (BBB) adjustment and observation could be performed [22], and the cerebral blood flow distribution could be tracked [23]. Lately, visible-NIR-II compatible skull optical clearing window was established, which was capable of achieving imaging depth of hundreds of microns in combination with NIR-II excited nonlinear optical microscopy [24]. Very recently, long-term skull optical clearing technique was developed to make the skull transparent over weeks [25,26].
This paper mainly focused on the study of the thrombolytic effect of UK on three kinds of cerebral vessels of different sizes. In the study, in vivo tissue optical clearing technique was introduced to establish a skull optical clearing window, through which light could be easily focused on a small area of mouse brain. Therefore, using thrombin-combined photothrombotic model, targeted ischemic stroke was established wherever needed through the intact skull. Herein, vascular occlusion was induced in distal small vessels of MCA, secondary branches of MCA, and MCA, respectively. Laser speckle contrast imaging [27,28] and TTC staining was performed to evaluate the targeting ability of the model. In addition, immunofluorescence imaging was performed to evaluate whether the thrombi contained both platelets and fibrin. Next, the blood flow changes in the three kinds of vessels after stroke were monitored through the skull optical clearing window for 24 hours to study the progression of embolization at these different sites. Furthermore, UK treatment was introduced after stroke, and the blood flow changes were also monitored to evaluate the therapeutic effect. Finally, quantitative analysis was performed to compare the outcome of UK treatment on the different vessels with different sizes. This work holds potential to provide reference for the selection of stroke treatment plan.
2. Material and methods
2.1 Animals
All animal procedures were approved by the Experimental Animal Management Ordinance of Hubei Province, China, and carried out in accordance with the guidelines for the humane care of animals. 8-week-old female BALB/c mice (∼20 g) were supplied by the Wuhan University Center for Animal Experiment (Wuhan, China) and housed and bred in Wuhan National Laboratory for Optoelectronics with a normal cycle (12 h light/dark). 45 mice were used to determine the light dose for photothrombosis establishment for three kinds of blood vessels (15 mice for distal MCA, 15 for secondary branches of MCA, and 15 mice for MCA. 5 mice for each light dose). 3 mice were used for immunofluorescent staining and confocal imaging to analyze thrombosis component. 30 mice were used to evaluate the thrombolysis effect of UK (10 mice for distal MCA, 10 for secondary branches of MCA, and 10 mice for MCA, where 5 mice for each experimental group and 5 mice for each control group).
2.2 Skull optical clearing procedure
The establishment of the skull optical clearing window was the same as we previously reported [23]. Two solutions were used for the skull optical clearing procedure. Solution 1 (S1) was a saturated supernatant of ethanol (Sinopharm, China) and urea (Sinopharm, China) with a volume to mass ratio of 10:3 and an ethanol volume ratio of 75%. Solution 2 (S2) was a highly concentrated solution of sodium dodecylbenzenesulfonate (SDBS, prepared via mixing NaOH solution and Dodecyl Benzene Sulphonic Acid). The hydroxyl groups of ethanol could make collagen dissolution [29]. Urea served as a common optical clearing agent due to its hydration effect [30]. Besides, it could enhance the permieability of ethanol and accelerate clearance. SDBS is used to extract lipids and make the skull uniform [31]. Briefly, the mice were firstly anesthetized with 10% urethane (8 mL/kg) via intraperitoneal injection. The head of the mouse was unhaired with depilatory cream and a midline incision was made on the scalp along the direction of the sagittal suture, after which the mouse head was fixed and the fascia on the skull was removed, and the surface of the skull was dried using clean compressed air. Next, S1 was dropped onto the exposed skull to make the skull immersed for 10 min, during which period a swab was used to gently rub the exposed skull to enhance the penetration. Then, S1 was removed, and S2 was applied to the same area for 5 min when the skull optical clearing window was established. For the establishment and observation of ischemic stroke in distal MCA and secondary branches of MCA, a circular skull optical clearing window with a diameter of 5 mm was constructed. For the establishment and observation of MAC ischemic stroke, a bilateral cortical skull optical clearing window was constructed.
2.3 Laser speckle contrast imaging system
A home-built laser speckle contrast imaging (LSCI) system was used to monitor the blood flow velocity changes in the cerebral vessels. A He-Ne laser beam (632.8 nm, 3 mW) successively passed through an optical attenuator and a beam expander (after which the diameter of the light spot was about 1.2 cm) and was used to illuminate the mouse brain. The backscattered light was collected through a stereomicroscope and was captured by a CCD (696 pixels×520 pixels). The exposure time of the CCD was set as 20 ms. 40 continuous frames were used to calculate a blood flow distribution image with laser speckle temporal contrast analysis method [32–34,21].
For imaging of distal MCA stroke and the secondary branch of MCA stroke, the imaging field of view was 4.42 mm×3.30 mm, therefore the imaging resolution was 4.42 mm/696 (3.30 mm/520) =6.35 μm. For imaging of MCA stroke, the imaging field of view was 8.84 mm×6.60 mm, therefore the imaging resolution was 8.84 mm/696 (6.60 mm/520) =12.7 μm.
2.4 Ischemic stroke modeling
5 mg/mL of Rose Bengal (Sigma-Aldrich, USA) in phosphate-buffer saline (PBS, 0.01 M, Sinopharm, China) and 16 U/mL of Thrombin (Thrombin, Bovine, Sigma-Aldrich, USA) in PBS (0.01 M, Sinopharm, China) were prepared for further use. It is worth mentioning that “U” is the unit of drug potency. Generally, the instructions will indicate the content as “U”. After establishing skull optical clearing window on mice, distal MCA and the secondary branch of MCA could be clearly observed. For MCA photothrombosis, surgical removal of the temporal muscle on the lateral side was performed to expose the MCA. Next, a mixture of Rose Bengal solution (100 μL) and Thrombin solution (100 μL) was intravenously injected, followed by immediate irradiation of a 532-nm laser onto the target blood vessels, respectively. The 532 nm wavelength was chosen because it was the absorption peak of photosensitizer Rose Bengal. The area of irradiation was also optically cleared over the MCA and secondary branches. Figure 1(A) shows the position of distal MCA, the secondary branch of MCA and MCA, respectively. Figure 1(B) illustrates the procedure of photothrombosis establishment on three kinds of cerebral vessels. For the modeling of stroke on distal MCA and secondary branches of MCA, the areas of 532-nm laser irradiation were under the optical clearing skull window. For modeling MCA stroke, when the muscle on the lateral side was removed, the large MCA could be observed under the S1 treated lateral skull. In this case, laser irradiation could be performed without removing the lateral skull.
Fig. 1. (A) Illustration of the positions of three kinds of cerebral vessels. The green circles indicate the locations of 532-nm laser irradiation for three kinds of vessels. Blue, cyan and violet dashed outlines indicate optical cleared locations for observing stroke of distal MCA, secondary branches of MCA and MCA, respectively. (B) Schemes of the procedure of photothrombosis establishment of three kinds of vessels. For distal MCA and secondary branches of MCA. photothrombosis, the mouse head was fixed by glued onto a sheetmetal with a hole. For MCA photothrombosis, the mouse head was fixed by using two sticks against the ears and a clip over the mouth. (C) Schemes of the procedure using LSCI to track the blood flow velocity changes before and after photothrombosis establishment, as well as after urokinase (UK) treatment.
To determine the proper dose for embolization of vessels with different sizes, a series of experiments were performed. For distal MCA photothrombosis, irradiation doses of 5 mW×30 s, 5 mW×1 min and 5 mW×2 min were introduced, respectively. For secondary branch of MCA photothrombosis, irradiation doses of 5 mW×1 min, 5 mW×3 min and 5 mW×5 min were introduced, respectively. For MCA photothrombosis, irradiation doses of 20 mW×1 min, 5 mW×3 min and 5 mW×5 min were introduced, respectively. After irradiation, the LSCI laser system was used to evaluate whether the embolus was formed in the target vessels.
2.5 Urokinase treatment
104 U of urokinase was dissolved in 1 mL of 0.9% normal saline, and the solution was stored at 4° for further use. After the successful establishment of the thrombosis model, the urokinase solution (500 U/g [15]) was intravenously injected into the mouse body. The cerebral blood flow before/after photothrombosis establishment, and 0 min, 5 min, 15 min, 35 min, 60 min and 1 day after urokinase treatment was tracked using LSCI. The whole procedure of tracking the blood flow changes before and after urokinase treatment is shown in Fig. 1(C).
2.6 Immunofluorescence
Mice were perfused with PBS 15 min after photothrombosis establishment. The mice brains were removed and fixed in PBS with 2% paraformaldehyde (PFA) overnight at 4 ℃, and then fixed in 2% agarose, followed by slicing into 100 μm-thick sections using a vibratome (Leica VT1000, Germany). The brain sections were firstly washed 3 times (5 min for each) with wash solution (0.2% Triton-X-100 in PBS), secondly incubated in the blocking solution (a mixture of 2% Triton-X-100 and 5% normal goat serum in PBS) for 1 hour, followed by incubation with Anti-human Fibrinogen/AF555 antibody (1:200; bs-1240G-AF555, Beiijng Biosynthesis Biotechnology co. ltd. China) and Anti-Integrin alpha 2b/AF488 antibody (1:200; bs-2636R-AF488, Beiijng Biosynthesis Biotechnology co. ltd. China) overnight at 4°C in PBS containing 0.2% Triton-X-100 and 0.5% normal goat serum. Next, the samples were incubated at room temperature for 2 hours and then washed 3 times and were observed with a commercial laser scanning confocal microscope (LSM 710, Zeiss, Jena, Germany).
2.7 TTC staining
2,3,5-Triphenyltetrazolium chloride (TTC, Sigma-Aldrich, USA) was dissolved in PBS (wt/vol = 2%) and stored at 4 ℃. The mouse was sacrificed with cervical dislocation 24 hours after photo-thrombosis establishment. The whole brain was removed immediately and was put in the TTC solution for 15 min, during which period the normal tissue eventually became red while the infarction area was pale.
2.8 Statistical analysis
All data were analyzed by using Image J software that was developed by National Institutes of Health (Bethesda, MD, USA) and were performed manually on the raw image stacks. After the relative blood flow velocity distribution was quantified from the images, the Shapiro-Wilk (S-W) test was performed for normality. The quantified data were presented as mean ± s.d. for normally distributed data and as median ± interquartile range for data that do not conform to a normal distribution. independent-samples T test was used to reveal significant difference.
3. Results
3.1 Skull optical clearing window assisted photothrombosis model establishment
To evaluate the effect of UK on blood vessels with different sizes, first we investigated the proper light dose for each kind of blood vessel. As shown in Fig. 2, the target distal of MCA had a size of 68 ± 6 μm. Under the light dose of 5 mW×30 s, the blood flow in small vessels in the distal MCA was basically unchanged. Under 5 mW×1 min light dose, blood flow of target vessel decreased, but was still higher than surrounding background, and the vascular contours are still visible using LSCI. On the contrary, under the light dose of 5 mW×2 min, there was no significant difference between target blood flow signal and background signal around the target vessel. In addition, the TTC staining results showed the infarct area is as expected (Fig. 2(C)). Thus, 5 mW×2 min was determined to be just enough to form a thrombosis in the distal vessels of MCA and was used for further study on evaluation of UK.
Fig. 2. Light dose determination of photothrombosis on small vessel in distal MCA. Each mouse was monitored with LSCI before 532-nm light irradiation (as baseline) and after irradiation (as post-PT image). (A) Representative LSCI images of distal MCA before and after 532-nm laser irradiation with various doses. Color bar represents the signal intensity. (B) Quantitative analysis of blood flow velocity changes in the target vessels after irradiation. The ROIs of vessels were chosen as the white circles in (A), and the sur-rounding background was chosen as the red circles in (A). ***p < 0.001, and ns: no significant difference. n = 5 for each light dose. (C) Representative photo of brain after TTC staining. 24 hours after irradiation at light dose of 5 mW×2 min. The cyan dashed frame indicates the infarct area. PT: photothrombosis treatment.
As shown in Fig. 3, the targeted secondary branches of MCA had a size of 95 ± 7 μm. Under the light dose of 5 mW×1 min, the blood flow in secondary branches of MCA was basically unchanged. Under 5 mW×3 min light dose, blood flow of target vessel decreased, but was still higher than surrounding background, indicating the vessels were not completely clogged. Under the light dose of 5 mW×5 min, the target blood vessels disappeared under LSCI, and there was no significant difference between target blood flow signal and background signal around the target vessel. In addition, the TTC staining results showed the infarct area was larger than that of distal MCA occlusion, which was as expected (Fig. 3(C)). Thus, 5 mW×5 min was determined to be just enough to form a thrombosis in the secondary branches of MCA and was used for further study on evaluation of UK.
Fig. 3. Light dose determination of photothrombosis on secondary branches of MCA. Each mouse was monitored with LSCI before 532-nm light irradiation (as baseline) and after irradiation (as post-PT image). (A) Representative LSCI images of secondary branches of MCA before and after 532-nm laser irradiation with various doses. Color bar represents the signal intensity. (B) Quantitative analysis of blood flow velocity changes in the target vessels after irradiation. The ROIs of vessels were chosen as the white circles in (A), and the surrounding background was chosen as the red circles in (A). ***p < 0.001, and ns: no significant difference. n = 5 for each light dose. (C) Representative photo of brain after TTC staining. 24 hours after irradiation at light dose of 5 mW×5 min. The cyan dashed frame indicates the infarct area. PT: photothrombosis treatment.
For the evaluation of the blockage in MCA, as the MCA was on the side of skull, not in the imaging vision, it was impossible to observe the changes of the inside blood flow but could only reflect the embolism of MCA through the blood flow of its branches. As shown in Fig. 4, under the light dose of 20 mW×1 min, the blood flow in the branches of MCA was basically unchanged. Under 20 mW×3 min light dose, blood flow of the branches decreased, but was still higher than surrounding background, indicating the MCA was not completely clogged. Under the light dose of 20 mW×5 min, almost all the branches of MCA disappeared in the LSCI images, and there was no significant difference between target blood flow signal and background signal around the target vessels. In addition, the TTC staining results showed the infarct area is as expected (Fig. 4(C)) and almost the entire cortex of the hemispheres is infarcted. Therefore, 20 mW×5 min was determined to be just enough to form a thrombosis in the MCA and was used for further study on evaluation of UK. Compared to distal MCA and secondary branches of MCA, MCA needed much higher light dose to be occluded, which was reasonable because the size of the mouse MCA was 180-230 μm, which is much larger than that of those two kinds of vessels [36,37].
Fig. 4. Light dose determination of photothrombosis on MCA. Each mouse was monitored with LSCI before 532-nm light irradiation (as baseline) and after irradiation (as post-PT image). (A) Representative LSCI images of secondary branches of MCA before and after 532-nm laser irradiation with various doses. Color bar represents the signal intensity. (B) Quantitative analysis of blood flow ve-locity changes in the target vessels after irradiation. The ROIs of vessels were chosen as the white circles in (A), and the surrounding background was chosen as the red circles in (A). ***p < 0.001, and ns: no significant difference. n = 5 for each light dose. (C) Representative photo of brain after TTC staining. 24 hours after irradiation at light dose of 20 mW×5 min. The cyan dashed frame indicates the infarct area. PT: photothrombosis treatment.
3.2 Thrombosis component analysis
Figure 5 shows fluorescence microscopic imaging of brain slices of mice thrombin-combined photothrombosis establishment. As shown in Fig. 5(A), abundant signals of both Integrin alpha 2b (used to stain platelets) and Fibrinogen (used to stain fibrin) were observed in the light irradiated area while not observed in the contralateral area, indicating that both platelets and fibrin were formed under the laser irradiation. Moreover, as shown in Fig. 5(B), at high magnification, it was clearly observed the presence of both platelets and fibrin in the thrombus inside larger vessels and smaller vessels.
Fig. 5. Representative mouse brain slice immunofluorescence images after thrombin-combined photothrombosis establishment. (A) Representative confocal images under a 5× objective. (B) Zoomed views of the yellow and red frame areas in (A) (under a 60× objective).
Such results demonstrated that, under the skull optical clearing window, the thrombosis that consisted of both platelets and fibrin could be formed using the thrombin-combined photothrombosis model, which was consistent with that under the open-skull window [18]. Therefore, we were able to establish targeted ischemic stroke with the thrombosis composition more clinical than traditional photothrombosis when the brain was under the protection of the skull, thus under its original environment.
3.3 Comparison of the thrombolytic effect of urokinase on vessels of different sizes
As shown in Fig. 6(A), after photothrombosis establishment on distal MCA, the is-chemia would not recover itself during next 1-hour observation. In addition, the is-chemic area expanded further after 1 day. On the contrary, the blood perfusion rapidly increased with UK treatment, but started to decrease again within the 1-hour observation.
Fig. 6. Representative LSCI images at various time post ischemic stroke with/without UK treatment. The thrombin-combined photothrombosis was established at distal MCA (A), the secondary branches of MCA (B) and MCA (C). The white stars state the center position of the 532-nm laser spot (the diameter of the spot was 1.3 mm). The dark dots indicate the representative areas of blood vessels in the stroke area and were used for statistics and calculations in Fig. 7. PT-0 min: right after photothrombosis establishment. T-5 min: 5 min after treatment of UK.
As shown in Fig. 6(B), the thrombosis in the secondary branches of MCA would cause a larger region of ischemic stroke than that of small vessel in distal MCA, and it would not recover if no pharmacological intervention was performed. On the contrary, 15 min after UK treatment, most of the blood flow signals that had been missing were back to view. More importantly, the blood vessels did not become clogged again 1 day later.
As for MCA ischemic stroke, the blood flow signals of almost the entire cortex of the hemispheres were gone (Fig. 6(C)), and this continued 1 day later without UK treatment. as shown, UK treatment would work, but work more slowly than that of secondary branches and distal MCA. In addition, UK could not restore all the lost blood flow signals within an hour as in the first two cases. Furthermore, 1 day after stroke and treatment, only about half of the vessels recanalized.
We further quantitatively analyzed the blood flow velocity after UK treatment to evaluate its thrombolysis effect. The results are shown in Fig. 7. For the distal MCA vessels without the intervention, blood flow decreased to about 20% of baseline immediately after laser irradiation, then gradually decreased to 15% within 60 min and continued to decrease to 10% of the initial level 1 day later. If the mice were treated with UK, the mean value of blood flow recovered to 70% 5 min after the introduction of UK, and further recovered to 78% within 15 min, and continued to increase to 87% within 15∼35 min. However, the blood flow of the terminal vessels of the middle cerebral artery decreased again within 35∼60 min and decreased to 62% at 60 min. One day later, the blood flow signal of the terminal vessels of the middle cerebral artery was not visible, and the blood flow signal was about 6%.
Fig. 7. Quantitative analysis of blood flow changes after distal MCA stroke (A), secondary branches of MCA stroke (B) and MCA stroke (C). The thrombin-combined photothrombosis was established at distal MCA (A), the secondary branches of MCA (B) and MCA (C). PT: right after photothrombosis establishment. T-5 min: 5 min after treatment of UK. In the non-treated group, mice were monitored without UK treatment. 5 mice for distal MCA stroke; 5 mice for distal MCA stroke + UK treatment; 5 mice for MCA’s secondary branches stroke; 5 mice for MCA’s secondary branches stroke + UK treatment; 5 mice for MCA stroke; 5 mice for MCA stroke + UK treatment. For each mouse, we chose 5 area of blood vessels in the stroke area for relative blood flow velocity calculation.
For the secondary branches of MCA and their distal branches without intervention, the blood flow immediately decreased to 7.3% after irradiation, and the blood vessels were not visible at all. After that, the blood flow remained at a low level in the next 0-60 min and 1 day, and the relative value did not show significant changes. On the contrary, after the introduction of UK, the blood flow increased to 63% of the initial state at 5 min. At 15 minutes, the blood flow increased to twice the initial value, then gradually decreased, and recovered to the initial level at 35 min. After 35 to 60 min, the blood flow remained stable at about 110%, and a day later, the blood flow increased again to 174%.
For MCA stroke, the blood flow in its branches dropped to 14.7% immediately after the laser irradiation and remained nearly constant for the next 60 min or even a day later without UK treatment. In the UK-treated group, the the blood flow signal was still invisible, and the intensity of blood flow signal was maintained at 15%∼20% within the first 15 min after treatment. Then, the blood flow began to recover to 35% at 35 min, then continued to rise, reaching 40% at 60 min. One day later, the average blood flow intensity returned to 60% of its original level.
4. Discussion
In this study, the function recovery of the distal small vessels of the MCA, secondary branches of MCA, and MCA after UK treatment were tracked during 1 day with the combination of skull optical clearing window and LSCI technique. It was observed that, after ischemic stroke establishment on 3 kinds of vessels, the ischemic area will gradually diffuse over time without treatment, resulting in the further decrease or disappearance of blood flow signals around the core ischemic area. Studies have shown that, 1∼3 days after ischemia caused by traditional photothrombosis can be identified as the acute period of vascular thrombosis, in which period the brain cells in the infarct core will die first and the surrounding cells would get blood from other branches but become less active; then the core area of infarction will expand, and the symptoms of cerebral infarction will gradually worsen because the surrounding cells could not get enough supply for too long [38,39]. It could be assumed the reason why the infarct area of thrombin-combined photothrombosis model also further expanded after 1 day.
Our results showed that, with UK intervention, the blood flow in the distal MCA was quickly restored, indicating that thrombolysis occurred rapidly in such small vessels. However, the blood flow in the vessels dropped again after a period of time, and even disappeared in the observed area 1 day later, similar to that in untreated mice. This suggested that these small vessels undergo reinfarction after thrombolytic therapy with UK. In clinical practice, there are also cases of secondary blockage in the small arteries after thrombolytic therapy [40]. It is generally believed that the root cause of rethrombosis after thrombolytic therapy is the damage of vascular endothelial cells, resulting in increased platelet activity [41,42]. In our case, the mechanism of photothrombosis was to damage the vascular endothelial cells. Therefore, although blood flow was restored briefly, the wound surface of vascular endothelial cells still existed, which would continue to cause platelet aggregation here, and the small size of blood vessels was easy to cause secondary blockage.
For embolism of vessel with large blood vessel, such as MCA, blood flow began to recover 35 min after treatment, indicating that UK was slower to thrombolytic in larger vessels. In addition, it was observed that UK could not completely cure MCA embolism. 1 day after treatment, only the MCA branches in the parietal lobe recovered blood flow, while the MCA branches in the frontal lobe lost blood flow signals. Based on this, we assumed that, when the embolus busted, the fragments moved with blood flow to the branches. Since the frontal branch of MCA was relatively smaller, the fragments might clog the side branches. However, the parietal branch of MCA was larger, not easy to be blocked, therefore the embolus fragments could continue to dissolve in the movement.
This work also expands the application of in vivo skull optical clearing technique. The skull optical clearing windows have been mainly used for cortical neurovascular imaging in the first place [20,23]. Later, researchers began to perform optical manipulation (e.g. tissue ablation, BBB opening) through skull optical clearing windows [24,43,44], while such light-manipulation mainly have significance in biological study, with limited clinical relevance. This work, for the first time, utilized skull optical clearing window to establish a common clinical disease model, and used it for drug evaluation, therefore is of great significance to the in vivo optical clearing technique.
Besides thrombolysis effect, bleeding transformation is also important for a drug when used. In the future work, optical clearing window assisted thrombin-combined photothrombotic model will be also promising to study both the thrombolysis effect and the bleeding transformation of thrombolytic drug simultaneously, with the help of combined laser speckle imaging and fluorescent intravital microscopy [45].
5. Conclusion
The key to treating ischemic stroke is the timely recanalization of infarcted blood vessels. Intravenous thrombolysis is the main clinical thrombolytic method and the most effective treatment method. UK holds advantages of high thrombolysis effect, convenience of obtaining, especially long time window and low price. Therefore, despite its side effect is slightly stronger than atenolol enzymes, another fibrinolytic drugs, UK is still the common treatment in basic hospitals. In this work, ischemic stroke models on three kinds of cerebral vessels, such as distal small vessels of MCA, secondary branches of MCA and MCA, were established by combining skull optical clearing technique and thrombin-combined photothrombotic model to evaluate the treatment effect of UK on embolized vessels at different positions and with different sizes. The results indicated that, when UK was used to treat small vessels in the distal MCA, the block would occur again, and the treatment failed. When UK was used to treat secondary branches of MCA, the vessels were recanalized without secondary blockage and thrombolysis was successful. When UK was used to treat MCA, thrombolysis was slow and incomplete. Therefore, from our comparison, UK had the best effect on the secondary branches of the MCA. In the future work, it is valuable to investigate if higher dose of UK could help treat distal MCA and MCA. Such results would be meaningful in the clinical treatment of ischemic stroke. This work also provides a general tool for thrombolytic drug evaluation on targeted ischemic stroke on animals.
Funding
National Natural Science Foundation of China (61860206009, 81870934, 82001877, 81961138015); China Postdoctoral Science Foundation funded project (BX20190131, 2019M662633); Innovation Project of Optics Valley Laboratory (OVL2021BG011); Innovation Fund of WNLO.
Acknowledgments
The authors thank to the Optical Bioimaging Core Facility of WNLO-HUST for support in data acquisition.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this can be obtained from the authors upon reasonable request.
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