Endo UV Tech will describe the properties of UV dilation in reverse chronological order of discovery because clinical applications generate the most interest but are developed from basic research principles. Here we insert a caveat: although thrombectomy per se is applicable to arteries and veins, UV-induced dilation can be observed only in arteries because their walls are composed of nitrite-containing smooth muscle cells. As amply demonstrated, veins cannot be made to dilate by UV irradiation. But the most severe complications of venous thrombosis are expressed eventually as arterial occlusions, so the lack of a primary UV effect on veins is usually not a critical limitation. In the near future, we will study the extraction efficacy of thrombectomized clots enlodged in rabbit common carotid artery as augmented by UV laser dilation, or not. Histopathologic and immunostaining analysis will compare the residual arterial damage.
We investigated basilar artery (BA) dilation in canines with hemorrhagic stroke and subsequent BA vasospasm, for which there is no uniform clinical treatment. The UV laser method was applied in the form of endovascular irradiation induced by a ring beam emitted from a conical-tip optical fiber following saline flush. This configuration was able to reverse vasospasm quite consistently in the basilar artery of canines subjected to the double hemorrhage model (Fig. 1) despite being surrounded by blood injected into the cisterna magna, from where it diffused along adjacent arterial walls. Vasospasm is caused by scavenging of nitric oxide (NO) by free hemoglobin (released from lysed red blood cells), which then infiltrates the basal artery wall. Evidently the basal NO was replaced by NO produced anew, and in sufficient concentration, by UV laser photoscission of nitrites resident in the wall. Vasospasm reversal by this semi-local mechanism is unprecedented and cannot be duplicated pharmacologically (ref. 1). This study was an extension of US Patent No. 6,539,944 awarded on April 1, 2003. The semi-local nature is demonstrated by its transport across rather long distances (ca. 6 cm) in the vertebral and basilar arteries (ref. 1). Pharmacologically, this is ideal because the treatment is not systemic and is yet within its range of efficacy. The UV beam can dilate arterial segments that are nowhere near the point of irradiation. The NO can be produced at several uM concentration in the arterial wall, and is transferred along arteries by the chain process of nitrosation, leading to long-lived dilation and possibly dethrombosis (removal of aggregated platelets) from distal vasculature. In summary, sufficient NO is present in every mammal, and needs only to be released by UV light from its nitrite precursors.
Fig. 1. Top: Angiograms of the basilar artery (BA) from a sample dog at different time stages. Bottom left: proximal, medial, and distal segments marked (red lines) on the baseline angiogram. The location of these segments was kept constant for all cases. Bottom right: average diameters of the three segments at each stage; error bars represent standard deviation (n~9 each segment); S: statistically significant. Note the remarkable persistence of BA dilation on Day 5, 24 hr after UV laser irradiation. The study was financed by OpusGen LLC, Doral, FL. From ref. 1.
Fig. 2. Based on the canine findings, permission was granted in 2009 for a clinical trial of endovascular UV laser irradiation in Italy. This study was compromised at an early stage by lack of funding and disorganization by a 3rd party organizer, and the only data that survived is shown here. But it shows that this method does dilate an extra-cerebral artery in human, as required by our current goal.
Restoration of blood to starved brain regions is the central acute problem in ischemic stroke caused by an occlusive thrombus. Drugs to prevent formation of a platelet-rich arterial thrombus are available, but dissociation of existing platelet thrombi can be done only in clot models containing “seepage channels,” which conduct thrombin inhibitor drugs to the clot interior. Human occlusive clots do not have such channels, however, so thrombin inhibition in humans has not shown consistent benefit. Nonetheless, the concept of dethrombosis (dissociation of intraplatelet GPIIb-IIIa fibrinogen cross-links, and thus the thrombus) was deduced from this early work.
The results of tPA in human stroke are not uniform either. Because the thrombi are composed of alternating layers of platelets and fibrin, tPA access to fibrin is likely filtered through structural defects in the platelet layers, which slows the rate of tPA penetration into the thrombus. Embolic stroke following tPA treatment, or more often from a thrombus ejected from the heart, is inaccurately but most commonly simulated in animals by injecting fragments of blood clotted in air, which contain readily accessible fibrin. This model is used in thrombectomy research also.Unsatisfied with clots formed under static flow conditions, Watson et al designed a photochemical method for inducing occlusive thrombi in arteries. This can happen only if platelets dominate the occlusion process, because they can react much faster to vascular injury than fibrin can form. A patented photochemical method (U.S. Patent No. 5,056,006) based on the interaction of intravenous (i.v.) rose Bengal dye with a 562 nm argon-pumped dye laser yielded very efficient occlusion of rat middle cerebral artery due to florid endothelial damage (type II, singlet oxygen-mediated). Fibrin could be formed via endothelial damage achieved with complementary Type I photochemistry (473 nm argon laser and i.v.. flavin mononucleotide) mediated by triplet state free radicals, but fibrin could not be formed fast enough to occlude the artery during active flow. We maintain that no pure fibrin clot formation is possible in vivo in the absence of relative stasis.
Fig. 3. Top: Light microscopic view of distal MCA segment perfusion-fixed at 30 minutes, containing primary thrombus (braces) with proximal and distal extensions. Note distal erythrocyte cap (arrow). Lower left: TEM view of proximal end occlusion. Note predominant platelet composition and erythrocyte infiltration along arterial wall. Magnification x2400. Lower right: TEM view of distal end, showing sharply delineated border between the mass of aggregated platelets and the end cap composed of clumped erythrocytes. No fibrin is detected. Magnification x1620.
Reversal of thrombotic stroke caused by occlusive tPA-resistant platelet aggregates appeared unlikely until Watson became aware that ultraviolet light could relax smooth muscle cells in arterial segments by producing endothelial relaxing factor, later identified as nitric oxide (NO). It was soon found that rat middle cerebral artery irradiated with an external UV laser beam in vivo dilated very quickly (< 1 sec). Repeating this procedure with a severely constricted artery occluded by a platelet thrombus should create space for blood to bypass it, but remarkably the thrombus also dilated owing to formation and branching of multiple blood-conducting microchannels (Fig. 3A), concurrent with “unzipping” of the fibrinogen-interlocked platelet pseudopodia (Fig. 4C). This process is called UV laser-mediated dethrombosis (U.S. Patent No. 6,539,944). The NO acts as a small and readily diffusible thrombin inhibitor, regardless of tissue density. The accompanying ultraviolet laser-mediated arterial dilation (up to 300%) persists for many minutes and is even transmitted (by the chain process of protein nitrosation) proximally and distally, indicating the possibility of reperfusion benefit in remote microvascular locations (i.e, beyond recirculation).
UV irradiation of the platelet thrombus was begun proximally and continued sequentially to the proximal border of the long fibrin-containing secondary thrombus (top, Fig. 3), formed from the high concentration of platelet prothrombotic secretions. This secondary occlusion was weak and eventually disappeared due to the pressure head. We thought that the emboli released would continue to degrade into smaller fragments, but dethrombosis at 2 and 3 hr increased infarct volume. Thus, in our effort to reverse experimental stroke damage, we inadvertently observed for the first time reperfusion injury by emboli emitted from an untreated and unstable secondary thrombus. This may occur during thrombectomy, as the distal clot end seems similarly unstable.
Figure 4. Open cranial view of an occlusive platelet thrombus aged for 0.5 hour (A: primary thrombus site designated by arcs) and then (B) treated for 17 minutes with ca. 5 W/cm2 of 355-nm UV laser irradiation. C: Light microscopic characterization (by WD Dietrich) of segment partially recanalized by microchannels and penetrated by blood (arrowheads). UVT, final position of UV treatment beam; initial beam position was proximal to an occluded branch (left parenthesis in A) and was advanced sequentially toward it. Owing to vasodilation and dethrombosis (A, B) blood infiltrated even past the UVT position (upper region of C), demonstrating that this effect is semi-local. P denotes regions of compactly aggregated platelets remaining to be dethrombosed.
The thrombus of Figure 4 was examined at higher magnification (transmission electron microscopy) in order to determine details of microstructure. In general, intraplatelet binding by fibrinogen has been disabled by NO-induced inhibition of thrombin, and the platelets float freely. Accordingly, many microchannels have been formed into which blood has penetrated.
Figure 5. Transmission electron microscopic analysis (WD Dietrich, adapted from ref. 2) of recanalization in the thrombus of Figure 2C. A: Proximally, the interior channel borders are composed mostly of disengaged platelets with intact pseudopodia (arrowheads), but more distally, some platelet membranes appear indistinct (asterisk) in a region of highly compacted platelets. Many erythrocytes are seen, along with some leukocytes (thin arrows). Magnification ×1400. B: View of a distal channel showing partial or complete disappearance of platelet membranes (arrows) and exposure of their thrombogenic cytoplasm. Magnification ×7600. C: View of channel at lower right in A. Platelets have disengaged from their cross-channel neighbors by cleanly unraveling their pseudopodia (arrows). Magnification ×3150.
The contribution of fibrin to secondary thrombus instability (contrary to popular impression) was further investigated in Type II photothrombi aged 3 hr, instead of 30 min (Figs. 3-5).
Fig. 6. The primary platelet component at the green laser spot (A) was very hard at 3 hr and usually would not dissolve until sequential application of external UV laser irradiation for ca. 80 min. Then, and most interestingly, the secondary thrombus (B) would disappear owing to the pressure head as before, but would reoccur in fluctuating fashion (C,D) for as long as could be observed. The presence of fibrin here signified instability, as indicated by gaps in the structure (C, D).
Most remarkably, the primary thrombus (under laser irradiation location in A) would not recur. Apparently the initially adherent platelets do not respond to dethrombosis by thrombin inhibition, because they are bound instead to the damaged endothelium by von Willebrand factor. Therefore, to the extent that such adherent platelets remain to protect the endothelium, UV laser-induced dethrombosis in conjunction with thrombectomy promises to minimize arterial wall damage caused by mechanical friction during clot extraction. The density of the clot will decrease as well, thus enhancing permeability to stentriever emplacement. We conclude that mechanical damage CAN be mitigated and does not have to be accepted as inherent.
We suspect that augmentation of thrombectomy with an exogenous agent, if necessary, could be ideally augmented by applying UV laser-mediated dethrombosis first. The enhanced permeability resulting from the formation of multiple multichannels will permit such an agent to interact with the now platelet-unshielded fibrin layers. Certainly, emboli composed of undissolvable platelet aggregates as seen during current tPA therapy should be much reduced.
The table shows a phenomenon we have not yet published, which shows that rt-PA (rPA in the table) synergizes with UV laser microchannel formation to have a direct effect on reduction of recanalization time for both the primary platelet clot (not expected at all) and also the much weaker secondary clot. This has not been reported before, because pure platelet thrombi in arteries are so rarely studied. We invented the concept of recanalization time per unit length of artery because this best expresses the effectiveness of the sequential irradiation procedure we followed.
Watson BD and van Vurst H: U.S. Patent Application S.N. No. 17/508,833, “Device and Method for Dilation of a Tubular Anatomical Structure,” filed on October 22, 2021 and awarded as US Patent No. 11,446,089 on September 20, 2022.
Watson BD: U.S. Patent Application S.N. 09/592,610, “Dethrombosis Facilitated by Vasodilation,” filed provisionally as application No. 60/138,609 on June 11, 1999 and officially on June 12, 2000, and awarded as U.S. Patent No. 6,539,944 on April 1, 2003.
Watson BD: U.S. Patent Application S.N. 07/183,046, "Method for the Permanent Occlusion of Arteries,” filed April 18, 1988 and awarded as U.S. Patent No. 5,056,006 on October 1, 1991.
1 Watson BD, Sadasivan C, Hurst RW: Endovascular Ultraviolet Laser-Facilitated Reversal of Vasospasm Induced by Subarachnoid Hemorrhage in Canines. Acta Neurochir Suppl. 127, 127-138, 2020.
2 Watson BD, Prado R, Veloso A, Brunschwig JP, Dietrich WD: Cerebral blood restoration and reperfusion injury following ultraviolet laser-facilitated middle cerebral artery recanalization in rat thrombotic stroke. Stroke 33: 428-434, 2002.