Animal Models of Stroke Instigated by Photothrombosis and Mitigated by Dethrombosis

Investigator: Brant D. Watson, Ph.D., Professor of Neurology, Associate Professor of Biomedical  Engineering, Neuroscience Program, and Jacob Javits Neuroscience Investigator (NINDS), 1992-1999

Research Focus: Endovascular laser thrombus formation and recanalization in CNS vascular disease

Rationale:  Neurointerventions for stroke and vasospasm are mechanically disruptive and generate complications, such as emboli and inflammation, but recanalization by laser-mediated dethrombosis is specific and clinically feasible; its complement, photothrombotic occlusion, may find use in AVMs. 

Research Background:  Most stroke research utilizes high-throughput models of mechanically induced cerebral ischemia, which cannot accurately mimic a stroke caused by embolic or thrombotic occlusion of a cerebral artery.  We solved this modeling problem by inventing and patenting the technique of photothrombosis.  Here, an intense light beam interacts with a photosensitizing dye to induce formation of either a “white” (platelet) clot or a “red” (mixed-composition, fibrin-stabilized) clot in brain blood vessels.  We later invented and patented the method of UV laser-facilitated dethrombosis, which dissociates intraplatelet fibrinogen bonds, and restores blood flow through arteries occluded by white clots as the platelet matrix disappears.  Dethrombosis can also release  platelets from occlusive red clots in arteries to restore some flow, despite an extant fibrin network.  

Clinical Relevance:  In 2006, Marder et al. showed that human “red” clots of cardiac (e.g., A-fib) and “white” clots of intra-arterial (atheromatous) origin have the same structure: alternating layers of platelets and fibrin entrapping pools of red blood cells.  Platelet-specific dethrombosis should reperfuse this common structure either alone, or in combination with rtPA-mediated thrombolysis, because it greatly increases the rate of rtPA penetration through the platelets to the fibrin layers.

Photochemical Mechanisms and Devices for Producing Photothrombotic Stroke
Type I Photothrombosis

Type II Photothrombosis

Photosensitizing dye (flavin mononucleotide)

Blue-filtered arc lamp light, or laser irradiation with 458, 476 or 488 nm argon, or 473 nm Nd:YAG  

   Free radical-generating dye triplet state

                                    

   Chain endothelial peroxidation and vasodilation

  Mixed-composition aggregates with fibrin

    Red thrombus formation and vascular occlusion

Distal territory ischemia

Photosensitizing dye (rose Bengal, erythrosin B)

Green-filtered arc lamp light, or laser irradiation with 514.5 nm Ar+, 532 nm Nd:YAG, 543 nm HeNe, 562 nm Ar+/dye, 568 nm krypton

   Singlet oxygen (O21?g) generation via dye triplet state energy transfer

                                   

  Direct endothelial peroxidation and vasoconstriction

Platelet adhesion and aggregation

     White thrombus formation and vascular occlusion

   Distal territory ischemia

Note the complementary thrombotic responses elicited intravascularly by the Types I and II photochemical reactions. These are seen primarily in arteries, which require focused laser irradiation.  During diffuse irradiation of a cortical region (through the intact skull), another unusual complementarity is seen: the Type I process specifically occludes venous-side microvasculature, resulting in cortical petechial hemorrhage, while the Type II process occludes arteriolar-side microvessels, resulting in focal cortical infarction.  If the cortex is cooled to 34 oC, no platelet response to Type II damage is seen, but a lesion still develops from vasogenic edema (the development of which may be suppressed by a special irradiation geometry). 

With the apparatus at left, a cortical “ring” lesion defined by its locus of microvascular occlusion can be formed (note the ring-shaped laser beam on note card) on the cortical surface in rodents; cf. web page of collaborator R. A. deFazio.  The tissue within the ring lesion models stroke-in-evolution, because the ischemic annulus encroaches upon it radially as upon a penumbra, except that here the penumbra is inverted and its location reproducibly predetermined.  The laser (mounted at top) is a 532 nm mini ND:YAG), and can be used alone to create a “spot” locus of cortical ischemia, and cerebral artery occlusion if focused.  For occlusion of brain arteries in animals larger than rodents, more powerful lasers are usually needed. 
 

Animal Models of Stroke Instigated by Type II Photothrombosis
Rat Cortical Microvessel Photothrombosis
Middle Cerebral Artery Occlusion in Rat

Occluded arteriole in pia, perfused at 2 min; SEM x4160

Cortical “spot” lesion, carbon black view at 4 hr

“Thick ring” lesion,
carbon black view
at 2 hr
562 nm dye laser beam
positioned on dMCA
patent ipsilateral CCA)

Rose bengal dye-photosensitized dMCA thrombus

SEM (x265) of MCA platelet thrombus; note constriction (top right)
 
   Photothrombotic CCA Occlusion 
CCA lumen 70% stenosed  with platelets; SEM x400 SEM(x1800) of platelet embolus at 15 min Coronal section of 111 In-labeled
platelet embolus distribution (15 min)
Occlusive thrombus (X) in CCA via
2 nm  dye laser (Rh590) and
i.v. rose Bengal dye
 

Dethrombosis of Occlusive Photothrombi (Types I and II) in Rat Middle Cerebral Artery

Type I red thrombus (brackets) in rat dMCA at 1 hr after formation; distal (blanched) column is within dashes.

Type II platelet (white) thrombus  in rat dMCA at 0.5 hr; note severe vasoconstriction and blanching

Dilation of same thrombosed dMCA segment and thrombus via 355 nm Nd:YAG laser irradiation

dMCA at 1 hr after dethrombosis by 355 nm Nd:YAG laser light, 5 W/cm2.  Blood reflow is seen (no rtPA used).

Dethrombosis by UV laser is based on  photoscission of NO from adducts in SMC layers; NO then diffuses into the platelet matrix to disrupt GPIIb-IIIa fibrinogen cross links. Any thrombus can evidently be recanalized, and further, rethrombosis of the damaged lumen by platelets is suppressed.

Blood infiltration into pure platelet thrombus during UV laser dilation
Microchannel formation (    ) in  platelet network, TEM x 1400
 

Photothrombosis and Endovascular Dethrombosis of Rat Common Carotid Artery
To be useful interventionally in thromboembolic stroke, UV laser-facilitated dethrombosis must be delivered by the endovascular route.  A conical-tip optical fiber, ensheathed in a microcatheter, produces a ring-shaped laser beam which irradiates the inside of the arterial wall uniformly (left), resulting in immediate vasodilation (right) owing to NO release.  The beam is positioned proximal to the occlusion and thus avoids direct contact with it.  Frank dissolution of the dense platelet matrices seen in human thrombi is not possible with any current drug.  Only a locally produced gas (NO) at high concentration can diffuse throughout such a thrombus and recanalize the occluded segment (at bottom). This procedure works independently of rtPA, but if rtPA is needed, its efficiency against fibrin will be much increased.

Conical-tip optical fiber

Laser UV ring beam in water

Rat CCA before and after 1 min of 351 nm laser ring irradiation
 

X-ray Angiograms of Occluded and Recanalized CCA

Corresponding CCA Ultrasonogram
Rat CCA photothrombotic Type II occlusion aged for 2 hr;
a UV ring laser beam endovascularly facilitated recanalization.

Ultrasonic blood flow trace, acquired just distal to the irradiated LCCA segment,  showing platelet thrombus formation and its dissolution by dethrombosis.