2.1.

Monte Carlo Simulations

In this study, we employed previously developed Monte Carlo code^41,42 to simulate dose absorption maps for irradiation of skin and mucous tissue at the wavelengths of 405 and 660 nm and compare typical impact depths. Monte Carlo technique is based on the simulation of a large number of random photon walks in a given turbid medium with the specified optical properties and subsequent statistical analysis of the collected data.

The simulation was performed for uniform illumination with a total light dose of $50{\mathrm{J}/\mathrm{cm}}^2$ ($0.5{\mathrm{J}/\mathrm{mm}}^2$) at the surface that corresponded to the parameters of the regimes employed in animal and clinical studies. When describing the simulation results all the delivered doses are given in $\mathrm{J}/{\mathrm{mm}}^2$ and absorbed dose densities are given in $\mathrm{J}/{\mathrm{mm}}^3$ because the typical scale is of order of mm.

We considered a three-layer skin model, consisting of stratum corneum ($50\mu \mathrm{m}$), epidermis ($150\mu \mathrm{m}$), and dermis (3.8 mm), and a single layer model for mucous tissues. Tissue optical properties employed in Monte Carlo simulations are summarized in Table 1. The refractive index amounted 1.4 for all the layers.

Table 1

Typical optical properties of skin and mucous tissues at the wavelengths of 405 and 660 nm.43–46

2.2.

Animal Study

Morphological and functional changes in tissues as a result of PDT procedure were studied at the skin of a rabbit ear inner surface. The studies are approved by the Ethical Committee of the Nizhny Novgorod State Medical Academy (Protocol #13, 03.10.2017). Topically applied drug “Revixan” (Revixan Ltd., Russia) based on chlorin-type PS was employed in the study. The drug contains pure chlorin e6 in a concentration of 0.1% vol. The “Revixan” gel in the amount of 2 mL was administered to the rabbit ear inner surface with a cotton swab. In 20 min after application the rest of the gel was removed, as well, with a cotton swab. A laser at the wavelength of 405 nm (M-33A405-500-G, Shenzhen 91 Laser Co., Ltd., China) was employed as a light source; the total impact light dose was $50{\mathrm{J}/\mathrm{cm}}^2$. The dose was chosen in accordance with the typical doses employed in antimicrobial PDT (for example, see Refs. 4748.–49). The irradiance at the tissue area was tuned to $0.4{\mathrm{W}/\mathrm{cm}}^2$ resulting in total illumination time of 125 s. The treated area was $2\times 3{\mathrm{cm}}^2$, a total of six areas were treated. Optical monitoring of a PDT procedure was performed by OCT (OCT-1300E device, BioMedTech, IAP RAS, Nizhny Novgorod, Russia) and FI (fluorescence imaging device,⁵⁰ IAP RAS, Nizhny Novgorod, Russia). OCT imaging was performed before PS administration (intact tissue), in 15 min after PS administration, immediately after laser impact, in 1, 3, and 7 days after PDT procedure.

The tissue changes were verified morphologically. Biopsy samples for histological studies were taken in 1, 3, and 7 days after the PDT procedure. Biopsy samples were subjected to staining with hematoxylin-eosin. Morphological studies were performed employing Leica DM 2500 microscope. The following parameters were analyzed: signs of edema and inflammation and the area of the newly formed vessel. The degree of edema (percentage of edema lumen between tissue elements) and inflammation (the presence of neutrophils and lymphocytes) was evaluated by semiquantitative three-grade scale depending on the manifestation in histology images (criteria are shown in Table 2). Criteria for moderate and pronounced inflammation also include the presence of plethora, which is manifested by the excessive presence of erythrocytes in the dilated vessel lumens. For each score, an average over 10 fields of view was taken; a field of view size is $0.65\times 0.44\mathrm{mm}$ ($\times 400$ magnification).

Table 2

Semiquantitative scale for evaluation of the degree of edema and inflammation.

The area of the newly formed vessels lumen was calculated as the relative area of newly formed vessels lumens in histology images averaged over 10 fields of view. The newly formed vessels were distinguished in the histology images as small vessels with thin walls and young endotheliocytes. The variation of the area was expressed in percentage with respect to the control value before PDT procedure.

2.3.

Human Subject Study

Evaluation of the efficiency of antimicrobial PDT was performed in the frames of a clinical study. The study was approved by the Ethical Committee of Nizhny Novgorod State Medical Academy (Protocol #7, 03.07.2017) for studies with human subjects. All the patients signed an informed written consent. A total of eight patients with exacerbation of chronic pharyngitis were enrolled in the study and underwent antibacterial PDT treatment. For all the patients, a microbiology study was performed to determine the etiology of the diseases and OCT inspection of pharynx mucosa was made to evaluate its morphological and functional condition. Each patient underwent three PDT procedures performed daily (days 1, 2, and 3; day 1 is the day of the first PDT procedure); visual and OCT inspection of the treated area was performed after each procedure and then in 3 and 7 days after the first procedure (days 1, 2, 3, 4, and 8). PDT procedure was performed in the course of standard pharyngoscopy inspection with topically applied chlorin-based drug “Revixan” (Revixan Ltd., Russia) in the form of gel. The drug contains pure chlorin e6 in a concentration of 0.1% vol. In each patient, 2 mL of the “Revixan” gel was administered to the mucous membrane of the posterior pharyngeal wall with a cotton swab. In 20 min after application the rest of the gel was removed, also with a cotton swab. After removal, a laser impact was performed at the wavelength of 405 nm with the light dose of $50{\mathrm{J}/\mathrm{cm}}^2$. The radiation was delivered to pharynx through a special diffusor emitting average irradiance of $0.2{\mathrm{W}/\mathrm{cm}}^2$ at a distance of 1.5 cm from the diffusor surface. The irradiation time was 250 s and the approximate area of the irradiated pharynx sector was $4\times 5{\mathrm{cm}}^2$. Local anesthesia was not required for the PDT procedure. There were no patients with strong pharyngeal reflex enrolled in the study.

3.1.

Numerical Studies

The simulated absorption maps for planar irradiation at the wavelength of 405 nm for skin and mucous tissues are shown in Figs. 1(a) and 1(b), respectively. The local absorbed dose densities are given in J per ${\mathrm{mm}}^3$. Similar maps for the wavelength of 660 nm traditionally employed for PDT with chlorin-based PS are shown in Fig. 1(c) and 1(d) for comparison. The maps allow one to analyze the distribution of the absorbed dose over depth $Z$. For skin, the largest part of light energy is absorbed in the upper stratum corneum layer, whereas for mucosa the distribution is more uniform.

Fig. 1

Distribution maps of absorbed light dose density for (a, b) skin and (c, d) mucous tissue at a wavelength of (a, c) 405 nm and (b, d) 660 nm. Color encoding corresponds to the dose density in $\mathrm{J}/{\mathrm{mm}}^3$. The left panels demonstrate in-depth attenuation of local absorbed dose for 405 (blue line) and 660 (red line) nm.

To quantitatively characterize the part of the total incident light intensity ($0.5{\mathrm{J}/\mathrm{mm}}^2$) coming to the tissue surface, we built the dependence of the light intensity absorbed in the upper tissue layer on the layer thickness (Fig. 2). Unlike the absorption maps shown in Fig. 1, this dependence has a cumulative effect as it demonstrates the entire dose absorbed in the medium starting from the tissue surface down to the given depth $Z$, which is more critical in clinical applications where the dose delivered to the treated area should be evaluated. In the case of skin, the absorbed dose demonstrates a pronounced jump at small depths originating from the absorption in the stratum corneum layer; however, further increase in $Z$ demonstrates slow growth because the major intensity was absorbed or backscattered in the upper layers. Surprisingly, for skin, the dependencies for the considered wavelengths do not differ dramatically. On the contrary, the single-layer model for mucous tissue demonstrates more steady increase of the absorbed dose with an increase in the layer thickness; however, the total absorbed dose varies greatly due to significant difference in the absorption coefficients of mucosa at 405 and 660 nm, respectively. All the demonstrated dependencies show saturation character indicating that lower depth contributes insignificantly to the total absorbed dose. For skin, for both wavelengths the saturation values lie around $0.20{\mathrm{J}/\mathrm{mm}}^2$ indicating that about 40% of the incident energy is absorbed within tissue, whereas the obtained dependencies demonstrate depth distribution. For mucosa, the saturation value for 405 nm is about $0.28{\mathrm{J}/\mathrm{mm}}^2$ indicating that around 56% of the incident intensity is absorbed. This value is higher than that for skin due to high scattering in stratum corneum resulting in high backscatter from the tissue. For 660 nm, the absorbed energy is much smaller approaching $0.03{\mathrm{J}/\mathrm{mm}}^2$ (6% of the incident intensity) for the layer thickness of 4 mm.

Fig. 2

Absorbed light dose at wavelengths of 405 and 660 nm in the tissue upper layer of thickness $Z$ for (a) skin and (b) mucous tissue versus layer thickness.

3.2.

Animal Studies

In the animal study, accumulation of the PS within the tissue was confirmed by FI monitoring. Time evolution of the signals ratio at two excitation wavelengths indicated deeper penetration of PS into the tissue. The details of this approach to PDT monitoring are given in paper.⁴²

Visual inspection revealed no changes in the skin of the rabbit ear after the PDT procedure in the course of 8-day monitoring.

Typical OCT-images of different parts of rabbit ear are shown in Figs. 3(a) and 3(d). The upper layer with a total thickness of approximately $300\mu \mathrm{m}$ with moderate signal corresponds to skin consisting of epidermis and dermis, superficial epidermis is thin and can hardly be resolved in the OCT image. The lower dark layer with thickness of about $150\mu \mathrm{m}$ corresponds to cartilage and is surrounded by two layers with high OCT signal corresponding to perichondrium (30- to $50\text{-}\mu \mathrm{m}$ thick). Comparison of the intact tissue before and after drug application [Figs. 3(a) and 3(d) versus Figs. 3(b) and 3(e)] allows one to conclude that the drug acts as a clearing agent: it decreases scattering from the top tissue boundary and increases the signal from deeper layers. The gel, being the solvent of the PS, penetrates into the tissue resulting in refractive index matching, such as traditional clearing agents.⁵¹ Especially pronounced is the increase in signals from the perichondrium and cartilage layers. To characterize this effect quantitatively, the A-scans before and after drug administration were plotted at the same graph [Figs. 3(c) and 3(f)]. Comparison of the one-dimensional in-depth OCT signal profiles (A-scans) confirms visually observed changes: the peak from the top boundary disappears, whereas the signals from perichondrium and cartilage rise. Clearing effect can serve as an indicator of drug penetration and, hence, PS accumulation in tissue and OCT can be employed to monitor PS penetration dynamics in case of topical application.

Fig. 3

OCT images of intact skin of rabbit ear (a, d) before and (b, e) after drug topical administration and (c, f) corresponding A-scans.

In the course of OCT monitoring, dark elongated areas with the low level of OCT signal were observed after the PDT procedure (Fig. 4); this effect was the most pronounced in 3 days after the procedure [Fig. 4(c)]. In 7 days after the first procedure, this manifestation became significantly weaker [Fig. 4(d)]. We associate this effect with appearance of edema in the area of treatment. This assumption is in agreement with the results of histological studies.

Fig. 4

(Top) Angiography and (bottom) structural OCT-images of (a) rabbit ear before, (b) in 1 day, (c) 3 days, and (d) 7 days after PDT procedure.

Angiography images allow one to monitor the response of the tissue microcirculatory system to the PDT procedure. Visual evaluation shows that in 3 days after the PDT procedure, smaller vessels become more pronounced in the angiographic OCT images and the vessel net becomes denser. Indirectly, this may serve as an indication of activation of small vessels.

Histological studies revealed that in 1 day after the PDT procedure, moderate edema is observed accompanied by moderate lymphohistiocytic infiltration with single neutrophils. The vessels have ordinary structure and their concentration corresponds to normal histology of a rabbit ear [Fig. 5(a)].

Fig. 5

Histology images (hematoxylin and eosin staining) of rabbit ear tissue in (a) 1, (b) 3, and (c) 7 days after PDT procedure. The arrows indicate (a) edema manifestation, (b) vessel with thick wall, and (c) newly formed vessel with thin wall.

In 3 days after the PDT procedure, the histology inspection indicated the presence of a weak edema; however, the signs of inflammation were not detected. An increase in the area of newly formed vessel lumens in the histological images amounted 12% to 15%, which is associated with neoangiogenesis [Fig. 5(b)]. In 7 days after the PDT procedure, no edema or inflammation signs were revealed, whereas an increase in the area of the newly formed vessels reached 30% to 35% [Fig. 5(c)]. The results of the histological studies are summarized in Table 3.

Table 3

Manifestations in rabbit ear tissue after PDT procedure (histological studies).

3.3.

Clinical Studies

OCT inspection of the posterior pharyngeal wall mucosa allowed one to distinguish two morphologically different cases: hyperthrophic and atrophic pharyngitis. In case of hypertrophic pharyngitis [Figs. 6(a) and 6(b)], the epithelial layer (upper layer with moderate signal in OCT image) is thickened and the boundary between epithelium and lamina propria (layer with high OCT signal) is not distinct. In subepithelial layer (lower layer in the OCT image), the elongated zones with low signal level are detected which is typical for edema. In case of atrophic pharyngitis [Figs. 6(c) and 6(d)], the epithelial layer is significantly thinned and can be hardly distinguished above lamina propria. The revealed differences in the OCT images allow one to preliminary conclude on the ability of OCT for differential diagnostics of chronic pharyngitis due to distinguishing specific morphologic features.

Fig. 6

Typical OCT-images of pharynx with (a, b) hypertrophic pharyngitis and (c, d) atrophic pharyngitis.

The criterion for inclusion a patient into the OCT-monitoring group was the exacerbation of pharynx chronic inflammatory disease with the indication for PDT treatment. Microbiological examination of the smears from the posterior pharyngeal wall mucosa preceded the PDT procedure indicating the presence of diverse microorganisms: Staphylococcus haemolyticus, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, and other less common causative agents.

In the course of treatment, no complications or side effects of PDT were observed in the patients enrolled. The PDT procedure was well tolerated by all patients, no anesthesia was required; however, there were complaints of discomfort when applying PS to the posterior pharyngeal wall associated with different levels of pharyngeal reflex expression. After the PDT course, all the patients subjectively mentioned improved state of health.

Pharyngoscopy indicated the decrease of inflammatory processes (Fig. 7). In particular cases, hypervascularization of mucosa was visually noted [Fig. 7(c)]. Treatment efficiency was evaluated by patients’ subjective feelings (complaints), pharyngoscopy inspection, and results of microbiological studies. Microbiological studies in all patients after the PDT course have not revealed any pathogenic flora; however, in particular cases activation of saprophytic flora was detected.

Fig. 7

Photographs of the posterior pharyngeal wall mucosa with chronic inflammatory process: (a) day 1, before PDT procedure (congestive hyperemia, edema of the palatine arch, expressed vascular pattern), (b) day 2 (increase of edema and hyperemia), (c) day 4 (hypervascularization, abatement of inflammatory manifestations), and (d) day 8 (abatement of inflammatory manifestations, decrease of hypervascularization).

In six of eight patients positive changes were registered on the next day after the first PDT procedure (day 2), the patients noted stagnation of a discomfort in the throat, pharyngoscopy revealed local reaction manifested by hyperemia and contrasting of a vessel net. No progression of the local reactions was registered after the second and the third PDT procedures (days 2 and 3).

In one patient with atrophic pharyngitis, improvement was registered only after the full PDT course (three procedures), the patient noted decrease of dryness in the throat and sensation of a foreign body, pharyngoscopy revealed increase in vascularization of the posterior pharyngeal wall mucosa and decrease in the amount of mucous secretion at the posterior pharyngeal wall. Another patient with long-term pathology (several years) did not note any dynamics in his state; although, pharyngoscopy indicated positive dynamics: decrease in chronic hyperemia and edema.

To reveal morphologic and functional alterations in the course of PDT, the OCT monitoring of the posterior pharyngeal wall mucosa was performed. Typical OCT images obtained in the course of PDT monitoring are shown in Fig. 8. Initially, the upper epithelial layer is thickened [Fig. 8(a)], the dark elongated zone in lamina propria corresponds to the lymphatic vessel, elongated zone under lamina propria corresponds to the blood vessel; longer zones of the low signal indicate the presence of edema. After application to the posterior pharyngeal wall mucosa, the solvent acts as a clearing agent [Fig. 8(b)]. An increase in the imaging depth and the signal level may serve as a criterion of PS penetration speed and depth. After PDT procedure [Fig. 8(c)] the response of the lymphatic system is observed, manifested by the increase in the number of the elongated dark areas. Lamina propria becomes inhomogeneous and disorganized with long areas of smaller signal associated with edema as a reaction to the procedure. On the day 2 more pronounced response to treatment is observed, confirmed by the visual inspection [Fig. 8(d)]. The second PDT procedure was performed on the day 2; after it, the response is observed at wider area, in addition, the edema remains pronounced in and under lamina propria [Fig. 8(e)]. In 1 day after the third PDT procedure (day 4), the edema becomes less manifested [Fig. 8(f)], lamina propria becomes denser. In 5 days after the third PDT procedure (day 8) lamina propria remains dense, the signs of edema disappear. A decrease in epithelium thickness indicates disappearance of infiltration. Comparison of the OCT-image obtained in 7 days after the start of the PDT course reveals better condition of the posterior pharyngeal wall mucosa with respect to its initial condition [Fig. 8(a)] indicating the efficiency of the performed treatment.

Fig. 8

OCT images of the posterior pharyngeal wall mucosa with exacerbation of chronic inflammation: (a) day 1, before PDT procedure, (b) day 1, after PS application, (c) day 1, immediately after PDT procedure, (d) day 2, before the PDT procedure, (e) day 2, after PDT procedure, (f) day 4, and (g) day 8.