In vivo safety study using radiation at wavelengths and dosages relevant to intravascular imaging

Abstract. Significance Intravascular photoacoustic (IVPA) imaging can identify native lipid in atherosclerotic plaques in vivo. However, the large number of laser pulses required to produce 3D images is a safety concern that has not been fully addressed. Aim We aim to evaluate if irradiation at wavelengths and dosages relevant to IVPA imaging causes target vessel damage. Approach We irradiate the carotid artery of swine at one of several energy dosages using radiation at 1064 or 1720 nm and use histological evaluation by a pathologist to identify dose-dependent damage. Results Media necrosis was the only dose-dependent form of injury. Damage was present at a cumulative fluence of 50  J/cm2 when using 1720 nm light. Damage was more equivocally identified at 700  J/cm2 using 1064 nm. Conclusions In prior work, IVPA imaging of native lipid in swine has been successfully conducted below the damage thresholds identified. This indicates that it will be possible to use IVPA imaging in a clinical setting without damaging vessel tissue. Future work should determine if irradiation causes an increase in blood thrombogenicity and confirm whether damaged tissue will heal over longer time points.

The pre-trimmed carotid artery segments were processed in a series of graded alcohols and xylene and paraffin wax embedded. The resulting paraffin blocks were sectioned twice serially, with an effort to capture the center of each segment, at an approximate 5 µm thickness and mounted to slide. One slide was stained with hematoxylin and eosin (H&E) and the other with Gomori's Elastin Trichrome (GET). Microscopic evaluation of the resulting slides was conducted by Serge Rousselle, DVM, DACVP. Tabulated microscopic data are presented in Appendix A.
Morphology parameters were generally scored using the following semi-quantitative scale (0-4): Overwhelming feature; severe.

Score Description
Definitions and causes of damage types: Endothelial Cell Loss: Most sensitive endpoint. Endothelial cells are highly susceptible to any mechanical shear stress or thermal effect and are typically the first cell type to be lost.
Hypereosinophilic Smooth Muscle Cells: Acute cell injury in the media. The staining alteration indicates generally a change in cytoplasmic homeostasis. This can be an artifact of tissue handling or fixation if fixation conditions are not optimal or can be a very early sign of peracute mural damage. Interpretation is based on comparison between control and treated vessels as well as pattern and distribution of the change and associated changes (context).
Compressive/Pressure Necrosis/Cell Effacement (loss): Concentric change characterized by sheets of smooth muscle cells showing hypereosinophilia and pyknotic to karyorrhectic nuclei to cytolysis (loss of cellular features) leaving the vessel wall matrix intact. This change can be radial to circumferential and is always concentric (inside out).
Contraction bands: Alternating bands of hypereosinophilic and contracted smooth muscle cells alternating with pale hypocellular or acellular areas. This change indicates excessive vasoconstrictive stress and may lead to necrosis or regeneration.

Results & Discussion
Microscopic scoring of the local response of a total of forty eight (48) segments from 3 swine carotid arteries when treated with laser irradiation at several dosages showed the following:

General Perspective on Acute Vascular Changes Associated with Energy Delivery:
Energy delivery produce non-specific microscopic changes in tissues essentially characterized by degeneration and necrosis sometimes accompanied by evidence of extracellular protein matrix denaturation. In vessels, these changes target endothelial cells and media smooth muscle cells primarily. Extracellular matrix (ECM) can be affected by mechanical or energy delivery and cause laceration or tears (mechanical stress) or coagulation (radiative energy). Spurious factors can and frequently do complicate interpretation of vascular histology. Vascular histology can be significantly influenced by experimental conditions in-vivo and at tissue harvest (tissue handling and fixation). Catheterization of a target vessel alone can and typically erodes the endothelial surface through mechanical shear stress. Catheter bulk and relative rigidity can also impart localized compressive trauma sufficient to bruise the vessel wall and cause cell death in the media. This can be aggravated by vasospasm at the time of treatment. Tissue collection can also produce changes that mimic necrosis unless the tissue is fixed in-situ prior to dissection and excision/trimming. Tissue handling and pulling during dissection as well as trimming of target segments can also cause tissue damage that mimics compressive injury (crushing). Use of cauterizing scalpel can cause coagulation necrosis and denaturation of ECM.
Morphologically, the resulting effect on vascular cells include vacuolization, shrinkage and hypereosinophilia of the cytoplasm, pyknosis, localized cellular effacement or stretch tears in cellular sheets. Effect on ECM includes lacerations and coagulation/hyalinization (heat-induced denaturation). Interpreting the significance and cause of acute vascular changes must take into account the level of background noise as observed in concurrent untreated controls.
Microscopic Finding Ascribable to Treatment: In this study, the only change that was clearly dose-related consisted of media necrosis. This change is characterized by areas of media hypereosinophilia with pyknosis and/or karyorrhexis. It was observed at low severity in control segments (C0) and showed a clear increase in severity, incidence and circumferential extension at highest doses (C5 and C6). In group C3, there was a slight increase in incidence and circumferential extent that was of equivocal significance biologically (possible trend) considering the level of background noise in the controls. The changes observed at lower doses (C1, C4) were consistent with background noise.