Targeting blood-brain barrier leakage for visualizing , monitoring, and evaluating ischemia reperfusion injury with Rus-Tc99m Probe

Background : Cerebral ischemia-reperfusion (I/R) injury as a serious threat to human health is characterized by cerebral endothelial leakage, as a result of the damage of blood-brain barrier (BBB). It is thus quite attractive to realize real-time monitoring of BBB damage for therapeutic surveillance. Methods : In this study, a radioactive probe is constructed by conjugating ruscogenin (Rus), a neuroprotectants, to technetium-99m (Tc 99m ) to assess the damage of cerebral endothelial in BBB. Results : In vitro study proves that the probe can penetrate more eciently in damaged BBB. Then, longitudinal nuclear imaging distinguishes mice with BBB leakage from normal ones, which is validated by evans blue staining of brain tissue. Higher nuclear signal also correlates with poorer blood circulation in brain. Further, by visualizing brain signal during drug treatment, the probe nds that the most obvious protective ecacy of Rus occurs at 12 h post administration, which is superior than edaravone (Edara). Conclusion : Altogether, the probe is promising to monitor I/R injury real-time by radioactive-imaging of BBB integrity. Importantly, Rus as a neuroprotectants may serve as a potential theranostic agent for I/R treatment.


Introduction
Ischemic stroke has become the number one cause of death in China, and it is a serious threat to human health. The clinical methods and drugs for treating ischemic stroke are very limited. To date, tissueplasminogen activator (t-PA) is the only FDA-approved therapy for ischemic stroke. However, it usually exacerbates the disease through causing cerebral ischemia and reperfusion (I/R) injury [1][2][3] . I/R is characterized by a dynamic and complex pathophysiological process which preferably invade speci c organs including heart, brain, kidney and intestinal [4] . Meanwhile, it is usually multiple and resistant to conventional therapies, hindering successful surgical resection and drug treatment, and remains a bottleneck for sensitive detection and effective treatment in clinic [5,6] . As such, there is an urgent need to sensitively monitor the extent of damage to brain tissue by reperfusion and predict the development potential of stroke in the early stages. Therefore, it is particularly attractive to develop imaging methods with su cient molecular information to assess the risk of brain injury. As a promising candidate, single photon emission computed tomography (SPECT) imaging can sensitively and accurately identify molecular classi cations, avoiding optical quenchability and long-term instability. It is more convenient to use SPECT imaging for pharmacodynamic studies in animal models [7,8] .
Abundant evidence has con rmed that the destruction of the blood-brain barrier (BBB) is the primary factor in the pathogenesis of I/R injury [9,10] . Intravascular proteins and uids penetrate into the extracellular space of the brain parenchyma, leading to vasogenic brain edema and reducing blood ow to neurons, leading to irreversible apoptosis [11,12] . Some preclinical studies have proved that cerebral endothelial injury is the main sign of BBB destruction [13,14] . Intensive investigation of the cerebral endothelial is urgently needed. Previous studies have shown that ruscogenin (Rus) targets cerebral endothelial to inhibit NLRP3 activation and restrain NF-κB and ROCK/MLC pathways to improve I/Rinduced BBB damage [15,16] . Based on traditional pharmacokinetic analysis, the amount of Rus crossing the BBB is directly related to brain endothelium permeability. Such relatedness prompts that the method of cerebral endothelia-targeted Rus for both monitoring and evaluating drug e cacy could be developed, which is the motivation of exploiting novel image probe.
Technetium-99m (Tc 99m ) has often been used to label radiopharmaceuticals, due to its suitable physical and chemical properties, along with reasonable radionuclide cost. This is an important characteristic of thermosensitive compounds since they may be degraded during the labeling process. Several chelating agents are currently used to prepare stable complexes with Tc 99m , including ethylenediamine diacetate (EDDA), which requires mild labeling conditions and fast reaction rates. Importantly, compound labeled with Tc 99m and chelating agents have negligible toxicity and demonstrate obvious merits in comparison with iodine-containing molecules [17] . The appropriate half-life of Tc 99m enables easy visualize to mornitor speci c disease information for 24 h. In addition, SPECT combined with CT can be used as a structure and function detector for accurate assessment [18] . To date, the use of Tc 99m to target cerebral endothelial leakage for both damage imaging of BBB and drug e cacy assessments has rarely been reported.
In this study, an effective radioactive probe was developed using covalent conjugation of Tc 99m with Rus.
Rus, is a major effective steroidal sapogenina in Chinese herb that has been applied to treat acute and chronic in ammatory and cardiovascular diseases for years. It has also been widely used to treat chronic venous insu ciency and vasculitis in Europe for decades due to its ability to decrease capillary permeability and anti-elastase activity [19,20] . This probe (Rus-Tc 99m ) is designed to identify, monitor and assess the extent of BBB leakage. OGD/R and MCAO/R mouse models have been used to con rm the permeability to different levels of brain endothelium in vitro and vivo. At the same time, the probe can be used to evaluate the drugs (ruscogenin and edaravone) e cacy on cerebral endothelial damage.
Altogether, the probe is promising to evaluate I/R injury and evaluate drug e cacy in a real-time manner. Middle cerebral artery occlusion(MCAO) model A middle cerebral artery occlusion (MCAO) model was induced using the modi ed intraluminal lament method as follows: for blocking the blood supply to the ipsilateral hemisphere of the rat brain, the right middle cerebral artery was occluded by inserting a 4-0 nylon mono lament suture into the right internal carotid artery. The body temperature was sustained at 37℃ with a heated blanket throughout the procedure. The animals underwent 1h of MCAO and then were reperfused by careful withdrawal of the lament. In the sham group, an identical surgical procedure was performed without disturbing the arteries [21] .

Methods
Oxygen and glucose deprivation/reperfusion(OGD/R)model In order to generate I/R-like conditions by OGD/R in vitro, bEnd.3 cell was placed in a 37°C anaerobic chamber (0.2% O 2 , 5% CO 2 , 95% N 2 ) and cultured in glucose-free medium for 6h. After the oxygen-glucose deprivation, the cells were placed in glucose-containing DMEM with 10% FBS and then incubated under normoxic conditions for 24 h in order to affffect reperfusion. Control samples were taken from cells cultured under normal conditions [22] .

Tracer synthesis
Rus was labeled with Tc 99m using tricine and EDDA as the coligands. The radiolabeling procedure was . The vial containing the reaction mixture was sealed, cramped, and heated at 60℃ for 40 min. After being cooled to room temperature, a sample of the resulting solution was analyzed by radio-HPLC with HPLC method. The structure is shown in Figure 1. The synthesis of Rus-Tc 99m and its radiolabeling were achieved as mentioned.

TTC staining
Brains were quickly removed at 12 h post-ischemia. The 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining was performed to evaluate tissue viability and measure the infarct size. The infarct area was measured in NIH Image J software (Version 1.42; National Institutes of Health, Bethesda, Md). The infarct areas on each slice were summed and multiplied by slice thickness to give the infarct volume. Infarct volume was expressed as a percentage of infarction per ipsilateral hemisphere [23] .

Millicell assay
Cells were incubated in the Millicell bEnd.3 cells culture inserts in a humidi ed atmosphere of 5% CO 2 and 95% air for 7d. After OGD/R condition, the medium was removed. 100 μL of EB & Rus-Tc 99m solution were added into the Millicell cell culture inserts and 1 mL of Krebs buffer were added into the external chamber.
The cells were continuously incubated for another 60 min and then the external Krebs solution were collected and added to 96-well black microplate. EB concentration value was measured at an excitation wavelength of 620 nm, with an emission wavelength of 530 nm using an In nite M200 Pro plate reader (Tecan, NC, USA), radioactive counting value was measured by γ-ray counter.
Evans Blue (EB) in vivo BBB permeability was assessed by the leakage of EB stain into the brain following the tail-vein injection.
Two hours before the animals (n=6 for each group) were euthanized, 0.1 mL per 10 g body weight of 2% EB (Sigma Aldrich) in normal saline was injected into each animal. The animals were then anesthetized and perfused with normal saline. For the quantitative measurement of EB leakage, the ipsilateral hemisphere was removed and homogenized in 1 mL of trichloroacetic acid, then centrifuged at 12,000 g for 20 min. EB concentration was quantitatively determined by measuring the absorbance at 620 nm of the supernatant with a spectrophotometer. The EB content was quanti ed as micrograms of EB per gram of tissue, using a standard curve [24] .

CBF measurement
Cerebral blood ow (CBF) was measured using laser Doppler owmetry. A computer-controlled optical scanner directed a low-power laser beam over the exposed cortex. The scanner head was positioned parallel to the cerebral cortex at a distance of ~20 cm. A color-coded image indicating speci c relative perfusion levels was displayed on a video monitor. The images were acquired at the onset of ischemia and reperfusion 0, 6, 12 and 24 h (n=6 for each group) [25] .

Neurological de cits
Neurological de cits of the experimental animals were graded on an 18-point scale as previously described. The measurement of neurological de cits consisted of the following tests: spontaneous activity, symmetry of movements, symmetry of forelimbs, climbing, reaction to touch, and response to vibrissae touch. All six individual tests were scored on a four-point scale as 3, 2, 1, or 0. Final score was obtained by adding the scores recorded for each individual test, with a maximum score of 18 observed in healthy animals [26] .
In vivo SPECT/CT All in vivo imaging experiments were conducted on a dedicated small animal SPECT system (NanoSPECT/CT, Mediso) in Xiamen university, center for molecular imaging and translational medicine, calibrated for technetium-99m (Tc 99m ) emitted γ-radiation. Tracers were applied in amounts, corresponding to 10 MBq/animal intravenously into the tail vein 24 h before in vivo imaging. The mean scan time was about 60 min with an initial morphologic whole body spiral CT and a consecutive whole body SPECT scan with a frame time of 60 s. Animals were held under Iso urane inhalation anaesthesia (2% Iso urane in air) for the whole scan time. Data were reconstructed and analysed using in built VivoQuant Software and presented as percentage of the injected dose (%ID) per selected region of interest (ROI). For individual organ analysis, 3D ROIs were drawn on the anatomic CT images. The ROI size was identical for all parallel experiments. Mean values and standard deviation were calculated for %ID. After in vivo imaging, mice were either kept for longitudinal follow up examinations or sacri ced for tissue collection [7] .

Western blot analysis
The cells or samples (n = 6, for each group) were decapitated and rapidly collected. The prepared cells or tissues were homogenized in 1:10 (w/v) ice-cold protein extraction buffer in glass homogenizers. Soluble proteins were collected and centrifuged at 12,000 g for 10 min at 4℃, and then the supernatant was used to detect the level of ZO-1, occludin and the total protein. The protein concentration of the samples was determined by BCA protein assay reagent kit. Equal amount of protein lysate (50 μg) in each group was separated by 8% and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins on the gel were subsequently transferred onto a nitrocellulose membrane (260 mA, 2 h). The membranes were blocked with PBST containing 5% skim milk for 2 h at room temperature and then incubated with primary rabbit monoclonal antibody respectively overnight at 4℃(ZO-1, occludin, 1:800; Proteintech Group, USA). The membranes were then washed and incubated with secondary antibody (anti-rabbit IgG, 1:3000; Proteintech Group, USA) for 1.5 h at room temperature. The anti-actin antibody (1:1000; Proteintech Group, USA) served as control. The protein bands were visualized with enhanced chemiluminescence reagents (ECL), and the signal densitometry was quanti ed by an observer blinded to the groups of the animals using western blotting detection system (Quantity One, Bio-Rad Laboratories, USA) [28] .

Statistical analysis
All results are expressed as mean±standard deviation (SD). GraphPad Prism (GraphPad software, San Diego, CA, USA) was used for statistical analysis of the data. The significant of the differences between two groups was analysed using unpaired Student's t test, and multiple comparisons was performed by one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. All tests were two-tailed. P<0.05 was considered statistically significant.

Results
The characterization of Rus-Tc 99m The oxhydryl of Rus, Tricine, and the secondary amine of EDDA are coordination with the Tc 99m , which formed six coordination bonds. Rus-Tc 99m was synthesized and analyzed by HPLC. Due to the enhanced water solubility after the Tc 99m labeling, the peak time of Rus was delayed compared to the standard (Fig.1A-B).
In vitro targeting of Rus-Tc 99m probe to cerebral endothelial leakage To determine the Rus-Tc 99m characterization in advance, we detected EB and radioactive counting extravastion in vitro. The millicell assay was used to construct different reperfusion time points, and the correlation between different degrees of barrier damage and EB, Rus-Tc 99m leakage was observed. The results showed that with extension of reperfusion time, the leakage rates of EB and the radioactive counting of Rus-Tc 99m increase, there is a linear relationship between the EB and Rus-Tc 99m ( Fig.2A-E).
In vivo targeting of Rus-Tc 99m probe to BBB leakage MCAO mouse models were constructed at different reperfusion time points, and the correlation between different degrees of barrier destruction and EB, Rus-Tc 99m leakage was observed using EB stain and SPECT imaging experiments. The results showed that with the prolongation of reperfusion time, the leakage rates of EB and Rus-Tc 99m signi cantly increased, and reached the highest point at 12 hours of reperfusion, there was a linear relationship between the EB and Rus-Tc 99m (Fig.3A-E). Meanwhile, the results shown in Fig. 3B demonstrated that Rus-Tc 99m generally were speci cally delivered and distributed more in the brain, fewer in the lung, spleen and heart.

BBB leakage in accordance with blood ow in MCAO
In order to con rm the correlation between Rus and blood ow, Doppler owmeter was used to detect blood ow changes at different reperfusion time points, SPECT imaging was used to detect the radioactive intensity of Rus-Tc 99m in real time. The results showed that as the reperfusion time prolonged, the blood ow became stable, but the intensity of Rus-Tc 99m radioactivity gradually increased and reached the highest level after 12 hours of reperfusion ( Figure 4A, B). There was a negative correlation between blood ow and Rus-Tc 99m radioactivity ( Figure 4C).

Evaluation e cacy based on SPECT imaging of BBB leakage
The above results showed that Rus-Tc 99m probe was able to detect the extent of BBB leakage. Then treated with Rus (10 mg/kg) and Edara to observe the correlation between Rus-Tc 99m and BBB. As is shown in Scheme 1, diagram of this part study. First, Rus was analyzed by HPLC in blood and in brain tissue (Supporting Fig.S1). After administration of Rus and Edara, EB stain and SPECT imaging were used to detect the leakage of BBB at different time points after reperfusion in vivo. The results showed that after the administration of Rus and Edara, the leakage rate of EB gradually decreased with the increase of reperfusion time, signi cant increase in CBF (Supporting Fig.S2) and the radioactive intensity of Rus-Tc 99m gradually weakened. The above results indicated that Rus and Edara could restore the integrity of BBB (Fig.5A-B). At the same time, it could be seen from the results that Rus exerted its effectiveness earlier than Edara in the reperfusion stage (Fig.5C-D).
Therapeutic effect of Rus on cerebral I/R injury This study con rmed the effect of Rus on cerebral infarction volume, neurological score, ultrastructure of brain microvascular endothelial cell and the expression of tight junction protein (ZO-1, occludin) after 12 h of reperfusion in mice after cerebral ischemia. The results revealed that Rus obviously reduced the volume of cerebral infarction after cerebral ischemia for 1 h and reperfusion for 12 h (Fig.6A,B), which decreased the neurological score (Fig.6C). Besides, Rus also could increase the expression of ZO-1 and occludin ( Fig.7A-B). However, Edara was less effective than Rus at 12h of reperfusion.

Discussion
Recent studies have shown the importance of the BBB in the development of stroke [30,31] . BBB is a complex, multi-factor physiological environment. The BBB probe could detect the level of BBB permeability in real time, providing a basis for clinical monitoring of the development of stroke [32,33] .
Based on previous research, Rus could target cerebral endothelial to improve the BBB damage caused by I/R [15,16] . Therefore, in this study, we chose Rus-Tc 99m as a probe for in vivo monitoring using SPECT imaging. Our current research provided direct evidence that Rus-Tc 99m depended on the permeability of BBB and targeted brain tissues. Administration of Rus could reduce the leakage rate of BBB and restore the expression of tight junctions. These results suggested that Rus might have potential as an adjuvant diagnosis and treatment to ischemic stroke.
In order to clarify the correlation between Rus-Tc 99m and barriers, this study rst performed in vitro veri cation using small chamber EB and radiolabeling assay in vitro. The results showed that EB and radioactive intensity also differ with the increasing degrees of barrier permeability. The barrier permeability and leakage rate of EB raises the intensity of radioactivity also increased. Above results suggested that Rus-Tc 99m was positively related to the degree of barrier opening. In order to further explain the correlation between Rus-Tc 99m and the BBB, MCAO mouse models with varying degrees of BBB damage in vivo were constructed and con rmed by EB and SPECT imaging. The results showed that BBB permeability increased with prolonged reperfusion time, and radioactive intensity in the brain also increased with prolonged reperfusion time. In brief, the radioactive intensity of Rus-Tc 99m could represent the level of BBB permeability and play an important role in the clinical development of stroke.
Very importantly, we labeled Rus with Tc 99m to assess the damage of BBB in MCAO/R. Interestingly, Rus-Tc 99m accumulates in the ischemic hemisphere, which indicated that BBB was damaged during cerebral ischemia after reperfusion, and Rus might penetrate the ischemic brain tissue from the vascular space of the BBB. To further illustrate the correlation between the probe and the BBB, changes in blood ow were detected using a Doppler owmeter. The results showed that although there was a negative correlation between the radioactive intensity in the brain tissue and the blood ow, they all indicated the level of BBB penetration. In other words, Rus-Tc99m Probe is promising to monitor I/R injury real-time by radioactiveimaging of BBB integrity. It was worth mentioning that Rus-Tc 99m didn't rely on blood ow to enter brain tissue, but relied on the permeability of BBB to enter the lesion.
The above studies have all occurred under pathological conditions. In order to con rm the e cacy between Rus and Edara, this study administrated Rus-Tc 99m to observe the drug onset time in brain tissue during BBB recovery. EB results showed that Rus and Edara could restore the destruction of BBB, and SPECT imaging showed that during the BBB recovery, the radioactive intensity in the brain tissue gradually weakens. The above results indicated that the reduced permeability of BBB results in a reduction in the amount of Rus-Tc 99m entering the brain. Interestingly, it could be seen from the results that Rus exerted its effectiveness earlier than Edara's in 12 h after the reperfusion stage. Hence, Rus could intervene early in reperfusion injury, providing a guarantee for preventing further deterioration of the disease. Compared with Edara at 12 hours of reperfusion, Rus could signi cantly reduce the volume of cerebral infarction, recover the neurological score and repair tight junction as same as Fig. 6 and Fig. 7.
Our results indicate that Rus targets the cerebral endothelial cells and maintains the integrity of the BBB.
Rus-Tc 99m Probe has a targeted recognition effect on the degree of BBB leakage in cerebral I/R injury. Our ndings provide a novel strategy for the diagnosis and treatment of I/R stroke.

Conclusion
In this study, Rus-Tc 99m was developed with multiple capabilities to visualize, monitor and evaluate drug e cacy. The probe could accurately identify highly leaky cerebral endothelial and track their colonization in the brain by targeting the BBB. The molecular information revealed by Rus-Tc 99m provided an assessment of mouse BBB integrity. In particular, BBB damage increased with prolonged reperfusion, which explained the reason for the increased Rus-Tc 99m permeability. More importantly, we found that Rus targeted BBB to restore I/R-induced brain damage, while Rus-Tc 99m was able to evaluate the drug e cacy. Interestingly, the most obvious protective e cacy of Rus occurred at 12 h post administration, which was superior than Edara. The probe was promising to visualize and monitor I/R injury, accelerating that Rus would be applied in clinic.

Declarations
Ethics approval and consent to participate All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Consent for publication
Informed consent was obtained from all individual participants included in the study.

Availability of data and materials
Supporting information is available online.

Competing interests
All the authors declare no potential con ict of interest.   BBB leakage in accordance with blood ow A)The representative images of cerebral blood ow of ipsilateral cortex in MCAO/R group under different reperfusion time. The magnitude of CBF is represented by different colors, with blue to red denoting low to high. B) Using SPECT imaging to detect the distribution of Rus-Tc99m in brain. C) Semiquantitative determination of the ratios of the radioactive intensity (red line) and CBF (blue line) of brain. Data were presented as mean±SD (n=3).

Figure 5
In vivo BBB leakage with differently treatment. A) Evens-blue staining of the brain tissues from the mice injected with differently drugs. B) In vivo imaging of the mice intravenously injected with differently treated using Rus-Tc99m. C) Semiquantitative determination of the ratios of the radioactive intensity of brain injected with differently drugs. D) Semiquantitative determination of the evens blue extravastion of brain injected with differently drugs. Data were presented as mean±SD (n=3).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.