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Correspondence to:
William P.Fay,M.D.
University of Michigan Medical Center
7301 MSRB III,1150 W.Medical Center Drive Ann Arbor,Michigan
48109-0644 USA
T el.:+1 734 763 7838,Fax:+1 734 936 2641 E-mail:wfay@umich.edu
低回不已Received May 14,2004
Accepted after revision June 28,2004
伊利诺伊香槟Financial support: This work was supported by NIH grants HL65224 and HL57346 (WPF) and by
NIH T32 T raining Grant HL07853 (SMD).
to induce thrombus formation within the microcirculation, as described below (6).
Photochemical injury
mannyThis method involves intravenous administration of a photo-reactive substance, Ro Bengal, followed by illumination of an expod arterial gment with green light (540 nm) delivered from a xenon lamp equipped with a heat-absorbing filter (7, 8).Ro Bengal accumulates in the lipid bilayer of endothelial and other cells (9). Exposure of Ro Bengal to green light triggers a photochemical reaction that produces singlet oxygen and promotes the formation of other reactive oxygen species that damage the vascular endothelium and initiate thrombus forma-tion. The photochemical injury model appears to induce more subtle vascular injury than the ferric chloride model, and there-fore the time necessary to form an occlusive thrombus is generally longer with the former model than the latter. In our experience, approximately 40 minutes is required to form a completely occlusive thrombus in the carotid artery of C57BL/6J mice that receive 10 mg/kg Ro Bengal. The photochemical injury model has been employed by veral investigators to identify important roles for a variety of hemostatic and fibrino-
Figure 1:Ferric chloride carotid artery injury.The left carotid artery is visualized through a discting microscope (arrow points to animal’s head).An ultrasonic flow probe (0.5VB,T ransonic Systems) and a 1x2 mm piece of filter paper saturated with 10% ferric chloride are prent on the artery.After 3 minutes the filter paper is removed,saline is placed in the wound,and
blood flow monitoring is initiated.
Figure 2:Measurement of carotid
artery phasic blood flow velocity after ferric chloride injury.“Zero” on X-axis reprents time at which ferric chloride solution was removed from surface of
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artery.Complete occlusion occurred 9 min-utes later (arrow),a total of 12 minutes after initiation of vascular injury.
lytic factors in thrombus formation after vascular injury. Most investigators have studied thrombosis within a normal gment of the carotid or femoral artery. However, Eitzman et al. dem-onstrated that the photochemical injury method can be ud to target vascular injury and thrombosis to an atherosclerotic gment of the distal carotid artery, thereby producing a model more reflective of atherosclerotic plaque rupture and thrombo-sis (10).
Attention to veral factors is necessary in order to obtain reproducible results with the photochemical injury model. Ro Bengal should be administered to each animal by the same route (typically via the tail or jugular vein) and over the same period of time – usually as a bolus. The lar light source should be maintained at a constant distance, usually 5-6 cm, from the blood vesl. The time interval between Ro Bengal infusion and initiation of blood vesl illumination should remain constant. We accomplish this by illuminating the artery before the Ro Bengal is infud.
Venous thrombosis models Photochemical injury
This method is performed as described for the arterial injury method, except that the jugular vein is ill
uminated (11). The time required to form an occlusive thrombus is determined by monitoring blood flow with a miniature probe.
Figure 3:Inferior vena cava (IVC) thrombosis model.(A) Visualization of IVC through discting microscope (arrow points to animal’s head).(B) Suture ligation of IVC and side branch to induce venous stasis.(C) Cross-ction of IVC excid 6 days after ligation,demonstrating occlusive thrombus and infiltration of inflammatory cells into wall of vein and the thrombus (hematoxylin/eosin staining;mag.x 20).
Venous stasis
This model involves ligation of the inferior vena cava (IVC) to induce venous stasis and thrombosis (12). Mice weighing 20 to 30 grams are anesthetized by inhalation of isoflurane gas and a midline laparotomy is made. The small bowel is exteriorized and placed to the left of the animal. The IVC is expod by careful blunt disction while sterile saline is applied at regular intervals to the exteriorized bowel to prevent its desiccation.Non-reactive prolene suture (7-0) is looped around the IVC immediately caudal to the origin of the renal veins and ligated,
Figure 4:Blood collection from inferior vena cava (IVC).The surgically expod IVC (visualized through a discting micro-scope;arrow points to animal’s head) is cannulated at the level of the left renal vein with a 27
gauge needle.
T able 1:Selected anesthetic agents for murine surgical procedures.
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along with major side branches, to completely obstruct blood flow (Fig. 3). The laparotomy incision is clod in 2 layers with 4-0 or 5-0 non-reactive suture material and the mou is allowed to recover from anesthesia. Two-six days later the IVC is harvested. The weight of the IVC gment in mg is divided by the length of the IVC gment in cm to determine the amount of thrombus formed. The excid venous gment can be embed-ded in paraffin to enable histologic analysis. Another model of IVC thrombosis involves tightening 2 sutures parated by 0.7 cm for 20 minutes (13). The thrombus is removed, rind, dried overnight, and weighed.
Mechanical trauma
Pierangeli et al. described a model in which forceps were ud to deliver a standardized “pinch” (1500 g/mm2) to the surgical-ly expod femoral vein (14, 15). Rather than measuring blood flow and occlusion time, the investigators ud a miniature fiberoptic device to transilluminate the injured gment of vein while it was being visualized through a stereo microsope equipped with a clod circuit video system. Computer-assisted gray scale analysis was ud to determine thrombus area and the kinetics of thrombus growth and dissolution over a 50 minute period.
海淀实验Microvascular thrombosis models Epinephrine-collagen infusion
This method involves intravenous administration of a solution of epinephrine (60 µg/kg) and collagen (0.06-0.5 mg/kg), usually via the jugular vein (13, 16). The substances induce systemic platelet activation and obstruction of the microcircula-tion. The endpoint measured is death.
ceenIntravital microscopy
Several approaches have been reported for inducing thrombi in microvesls as they are visualized through a microscope,
considerable
T able 2:Summary of murine thrombosis/thrombolysis models.
改变英文thereby allowing real-time imaging of thrombus growth and dissolution. Denis et al. described a method in which ferric chloride is ud to induce thrombosis within menteric blood vesls of the small intestine (6). For this procedure, platelets are isolated from a donor mou and fluorescently labeled ex vivo. Another mou is anesthetized and the labeled platelets are injected into the tail vein. A midline abdominal incision is made and the mou is laid on its side on the platform of a fluores-cence microscope that is connected to a video camera with recorder. The small intestine is exteriorized and an arteriole (diameter 60-100µm) within the mentery is visualized. Thirty µl of ferric chloride solution (250 mM) is placed on the surface of the mentery overlying the visualized arteriole. Platelet deposition on the arteriolar wall, occlusive thrombus formation, and embolization of thrombi can be visualized, recorded, and quantified. Andre et al. extended this technique by utilizing computer software to continuously measure fluorescence inten-sity of injured menteric arterioles, thereby providing a more quantitative asssment of thrombus growth in real time (17).
Ron et al. ud 2 forms of lar injury to study thrombo-sis within small veins of the mou ear (18). Direct lar injury of veins with diameters ranging from 100-350 µm can be induced by applying short bursts of a high-energy argon-ion lar to the luminal surface of the blood vesl. Alternatively,
Ro Bengal can be injected intravenously and photochemical injury and thrombosis can be triggered by focusing the lar on a vein. Video recordings of the evolving thrombus are captured, digitized, and analyzed with a computer software package to measure thrombus area and integrated optical density every few conds. Interestingly, analys of the effects of coagulation and platelet inhibitors on thrombus growth suggested that the 2 forms of lar injury induced thrombosis by different mecha-nisms. Whereas thrombi induced by direct lar injury appeared to involve mainly platelet interactions, thrombosis triggered by photochemical injury appeared to depend on both platelet inter-actions and activation of the coagulation cascade.
Falati et al. ud confocal and widefield microscopy to study thrombus formation within cremasteric muscle arterioles (diameter 30-60 µm) (19). A puld nitrogen dye lar (440 nm) is ud to induce vascular injury. Thrombus growth and disso-lution is imaged, recorded, and analyzed with a digital camera and computer workstation. As a component of this model, the investigators infu into mice fluorescent antibodies that are specific for tissue factor, fibrin, and platelets to enable real-time measurement not only of thrombus size, but also specific throm-bus components.
Thrombolysis models
Several approaches have been ud to study clot lysis in the living mou. Carmeliet described a method in which plasma clots containing 125I-fibrin are formed in vitro and injected into the jugular vein of an anesthetized mou (20). The clot embolizes and lodges within the pulmonary vasculature, where it gradually lys. Several hours later the animal is euthanized. The heart and lungs are removed en bloc and their radioactivity (counts per minute) is measured. Since the radioactivity of the clot is measured before injection, % clot lysis can be measured. Bdeir et al. described a related method in which radiolabeled fibrin microparticles are formed in vitro and injected into the tail vein (21). Pharmacologically-induced arterial thrombolysis can also be studied in mice (5). An occlusive thrombus is induced in the carotid artery by ferric chloride administration. The time required to achieve reperfusion after administration of a plas-minogen activator (PA) is measured. Since murine plasminogen is relatively resistant to activation by human and bacterial PAs (e.g. human tissue-type plasminogen activator and streptoki-na, respectively) purified human plasminogen is administered along with the PA in order to temporarily “humanize” the murine plasminogen activation system. Transgenic mice expressing human plasminogen could be ud to obviate the need for administering purified human plasminogen when studying pharmacologic thrombolysis.
Anesthesia
Mice must be anesthetized for periods ranging from minutes to hours in order to perform thrombosis protocols. Tracheal intu-bation and mechanical ventilation is not required for the models described in this review. While invasive (e.g. femoral artery catheter) or non-invasive (e.g. tail blood pressure cuff) moni-toring of systemic blood pressure during surgery might be desirable for some experiments, it is generally not performed. Anesthetic agents are administered to mice either parentally or by inhalation (Table 1). Parenteral anesthetic agents can be delivered by subcutaneous, intraperitoneal, or intravenous injection. Intramuscular injections should not be given to mice. Inhalation agents are administered either within a chamber or by a facemask. The former approach involves placing anesthetic-soaked cotton or gauze within an airtight container. The mou is placed within the container (but not in direct contact with the anesthetic) and removed after it becomes unresponsive. This approach is suitable only for short-term procedures, such as phlebotomy, since the mou recovers quickly as it breathes room air. For a longer surgical procedure, inhalation anesthesia with agents such as isoflurane or halothane must be adminis-tered with supplemental oxygen via a precision vaporizer and facemask. Inhalation anesthesia with isoflurane is the preferred method of anesthesia for mou surgical procedures. Adjunctive agents can be administered along with the anesthetic. For example, atropine (0.05 mg/kg) can be administered by sub-cutaneous injection as a preanesthetic agent to prevent the decrea in heart r
ate and excessive salivation that some anes-thetics can induce. Becau it has a relatively large body surface area/mass ratio, the mou is prone to hypothermia while under general anesthesia. To minimize body heat loss, the mou
