Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.
Stroke forskning har tålt mange tilbakeslag i å oversette nevro terapi i klinisk praksis. I motsetning til den virkelige verden terapi (Pa trombolyse) produserer sjelden fordeler i mekaniske okklusjon-baserte eksperimentelle modeller, som dominerer preklinisk hjerneslag forskning. Denne fordelingen mellom benk og nattbord antyder behovet for å ansette Pa-responsive modeller i preklinisk hjerneslag forskning. For dette formål, er en enkel og tPA-reaktive trombotisk slag modell oppfunnet og beskrevet her. Denne modellen består av transient okklusjon av den ensidige arteria carotis communis og levering av 7,5% oksygen gjennom en ansiktsmaske i voksen mus i 30 minutter, under opprettholdelse av dyret rektal temperatur på 37,5 ± 0,5 ° C. Selv reversibel ligering av den ensidige carotid arterie eller hypoksi hver trykkes cerebral blodstrøm bare forbigående, er kombinasjonen av begge fornærmelser forårsaket varig reperfusjon underskudd, fibrin og blodplateavleiring, og store INFARct i midten cerebral arterie-leverte territorium. Viktigere, haleveneinjeksjon av rekombinant tPA ved 0,5, 1, eller fire timer etter Thi (10 mg / kg) gitt tidsavhengig reduksjon av dødelighet og infarktstørrelsen. Denne nye slagmodell, er enkel og kan standardiseres tvers laboratorier for å sammenligne eksperimentelle resultater. Videre induserer det trombose uten craniectomy eller innføre forhånds dannet emboli. Gitt disse unike fordeler, er thi modellen et nyttig tillegg til repertoaret av preklinisk hjerneslag forskning.
Thrombolysis and recanalization is the most effective therapy of acute ischemic stroke in clinical practice1. Yet, the majority of preclinical neuroprotection research was performed in a transient mechanic obstruction model (intraluminal suture middle cerebral artery occlusion) that produces rapid recovery of cerebral blood flow upon removal of the vascular occlusion and shows little to no benefits by tPA thrombolysis. It has been suggested that the dubious choice of stroke models contributed, at least in part, to the difficulty in translating neuroprotective therapy to patients2,3. Hence, there is an increasing call for employing tPA-responsive thromboembolic stroke models in preclinical research, but such models also have technical problems (see Discussion)4-7. Here we describe a new thrombotic stroke model based on unilateral transient hypoxic-ischemic (tHI) insult and its responses to intravenous tPA therapy8.
The tHI stroke model was developed based on the Levine procedure (permanent ligation of the unilateral common carotid artery followed by exposure to transient hypoxia in a chamber) that was invented for experiments with adult rats in 19609. The original Levine procedure faded into obscurity because it only produced variable brain damage, but the same insult caused consistent neuropathology in rodent pups when it was re-introduced by Robert Vannucci and his colleagues as a model of neonatal hypoxic-ischemic encephalopathy (HIE) in 198110. In recent years, some investigators re-adapted the Levine-Vannucci model to adult mice by adjusting the temperature in the hypoxic chamber11. It is plausible that the inconsistent brain lesions in the original Levine procedure may arise from fluctuating body temperatures of adult rodents in the hypoxic chamber. To test this hypothesis, we modified the Levine procedure by administering hypoxic gas through a facemask, while maintaining the rodent core temperature at 37 °C on the surgical table12. As expected, stringent body temperature control greatly increased the reproducibility of HI-induced brain pathology. The HI insult also triggers coagulation, autophagy, and gray- and white-matter injury13. Other investigators have also used the HI model to investigate post-stroke inflammatory responses14.
A unique feature of the HI stroke model is that it closely follows the Virchow’s triad of thrombus formation, including the stasis of blood flow, endothelial injury (e.g. due to HI-induced oxidative stress), and hypercoagulability (HI-induced platelet activation) (Figure 1A)15. As such, the HI model may capture some pathophysiological mechanisms relevant to real-world ischemic stroke. With this idea in mind, we further refined the HI model with reversible ligation of the unilateral common carotid artery (therefore to create a transient HI insult), and tested its responses to tPA thrombolysis with or without Edaravone. Edaravone is a free radical scavenger already approved in Japan to treat ischemic stroke within 24 hr of onset9. Our experiments showed that as brief as 30 min transient HI triggers thrombotic infarction, and that combined tPA-Edaravone treatment confers synergistic benefits8. Here we describe detailed surgical procedures and methodological considerations of the tHI model, which can be used to optimize reperfusion treatments of acute ischemic stroke.
Stroke is a major health issue of growing significance for any society with an aging population. Globally, stroke is the second-leading cause of death with an estimated 5.9 million fatal events in 2010, equivalent to 11.1% of all deaths18. Stroke is also the third-leading cause of disability-adjusted life years (DALYs) lost globally in 2010, rising from the fifth position in 199019. These epidemiological data highlight the need of more effective therapies of acute (ischemic) stroke. However, despite…
The authors have nothing to disclose.
This study was supported by the NIH grant NS074559 (to C. K.). We thank all collaborators who contributed to our research articles that the present methodology report is based upon.
adult male mice | Charles River | C57BL/6 | 10~13 weeks old (22~30 g) |
Mobile Laboratory Animal Anesthesia System | VetEquip | 901807 | anesthesia |
Medical air (Compressed) air tank | Airgas | UN1002 | anesthesia |
Isoflurane | Piramal Healthcare | NDC 66794-013-25 | anesthesia |
Multi-Station Lab Animal AnesthesiaSystem | Surgivet | V703501 | hypoxia system |
7.5% O2 balanced by 92.5% N2 tank | Airgas | UN1956 | hypoxia system |
Temperature Controller with heating lamp | Cole Parmer | EW-89000-10 | temperature controllers |
Rectal probe | Cole Parmer | NCI-00141PG | temperature controllers |
Dissecting microscope | Olympus | SZ40 | surgical setup |
Heat pump with warming pad | Gaymar | TP700 | surgical setup |
Fine curved forceps (serrated) | FST | 11370-31 | surgical instrument |
Fine curved forceps (smooth) | FST | 11373-12 | surgical instrument |
micro scissors | FST | 15000-03 | surgical instrument |
micro needle holders | FST | 12060-01 | surgical instrument |
Halsted-Mosquito hemostats | FST | 13008-12 | surgical instrument |
5-0 silk suture | Harvard Apparatus | 624143 | surgical supplies |
4-0 Nylon monofilament suture | LOOK | 766B | surgical supplies |
Tissue glue | Abbott Laboratories | NC9855218 | surgical supplies |
Puralube Vet ointment | Fisher | NC0138063 | eye dryness prevention |
MoorFLPI-2 blood flow imager | Moor | 780-nm laser source | Laser Speckle Contrast Imaging |
Mannitol | Sigma | M4125 | in-vivo TTC |
2,3,5-triphenyltetrazolium chloride (TTC) | Sigma | T8877 | in-vivo TTC |
Vibratome | Stoelting | 51425 | brain section for in-vivo TTC |
Digital microscope | Dino-Lite | AM2111 | whole-braina imaging |
O.C.T compound | Sakura Finetek | 4583 | |
goat anti-rabbit Alexa Fluro 488 | Invitrogen | A11008 | Immunohistochemistry |
Cryostat | Vibratome | ultrapro 5000 | brain section for IHC |
Evans blue | Sigma | E2129 | Detecting vascular perfusion |
Microtome | Electron Microscopy Sciences | 5000 | brain section for histology |
Avertin (2, 2, 2-Tribromoethanol) | Sigma | T48402 | euthanasia |
Fluorescent microscope | Olympus | DP73 |