CN116710177A - Apparatus and method for applying photo-biological conditioning - Google Patents

Abstract

The present invention relates to an apparatus for applying photo-biological modulation (PBM) to a biological object, the apparatus comprising a light source delivering light at an appropriate temporal evolution of its optical power, the apparatus further comprising a processing and/or light control unit determining the appropriate temporal evolution of the optical power based on the light delivery geometry on/in the biological object and the biological object optical coefficients, characterized in that in each part of the volume of the biological object the PBM effect is caused continuously by generating one or more specific fluence rates during one or more specific times; in the following parametersThe fluence rate and the time of the specific combination are selected from the group: at 180+Period 3 of 30s+2mW/cm ² Or at 80+25s period 11+9mW/cm ² Or at 40+20s period 16+10mW/cm ² Or at 15+Period of 10s 25+10mW/cm ² Or at 40+Period 10 of 1s+9,7mW/cm ² . The invention also relates to different methods for application to a biological object (PBM), comprising the light source delivering light at a suitable temporal evolution of its optical power as described above. The present invention also relates to the above-described devices and methods for using PBM, optionally in combination with exogenous agents that participate in or regulate metabolism. The present invention also relates to the above-described devices and methods for applying PBM, optionally in combination with probes for monitoring metabolic activity occurring in a biological subject. Such monitoring can define optimal PBM light application conditions according to the following aspects: i) Time relative to metabolic activity, ii) fluence rate, and iii) duration of irradiation.

Description

Apparatus and method for applying photo-biological conditioning

Corresponding application

The present application claims priority from international application PCT/EP2021059842 filed on 1 month 10 in 2020, and filed on 15 months 4 in 2021, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present application relates generally to photo bio-modulation (PBM) and more particularly to an apparatus and method for applying photo bio-modulation therapy (PBMT).

Background

Definition of the definition

The following definitions apply herein.

-light: electromagnetic radiation having a wavelength in the range of 250nm to 3 μm.

Irradiance or principal incidence (primary incidence) E [ W/m ] ² ]: irradiance describes the power per unit surface received directly from a source.

Emissivity LW/(m) ² .sr)]: emissivity is the power of light emitted through or from a unit surface area and propagating within a unit solid angle in a given direction.

Fluence rate FW/m ² ]: fluence rate is the power into a sphere exhibiting a unit cross section. Which takes into account diffusion and/or scattering effects in the target environment. Fluence rates were measured with an isotropic power meter. Which takes into account the direct flux (irradiance) and the scattering and diffusion contributions. As with fluence (see below), this term is of fundamental importance in dosimetry where multiple scattering and diffusion in the target tissue are important.

Fluence or light dose ψ [ J/m ] ² ]: is the time integral of the fluence rate. Thus, fluence is the energy that enters a sphere exhibiting a unit cross section.

Absorption coefficient mu _a [m ^-1 ]: reciprocal of mean free path prior to photon absorption

Scattering coefficient mu _s [m ^-1 ]: reciprocal of mean free path between photon scattering

Reduced scattering coefficient mu _s '[m ^-1 ]：μ _s '＝μs(1-g)

-anisotropy factor g-: g is equal to the average of cos "theta", where "theta" is the deflection angle of the photons scattered by the particle.

Effective attenuation coefficient mu _eff [m ^-1 ]：μ _eff ＝(3μ _a (μ _a +μ _s ')) ^1/2

Photo-bioregulation, referred to herein as PBM, refers to the treatment of a biological object (e.g., a tissue or organ) with light of a specific wavelength. Such treatment may promote regeneration and remodeling of tissues or nerves, eliminate inflammation, alleviate edema, relieve pain, regulate the immune system and metabolism. It has a positive effect on age-related macular degeneration, blood treatment, wound healing, immunomodulation and possibly even on viral and bacterial infections.

Many conditions are associated with disturbances of metabolism, including defects in mitochondrial respiration. These conditions include chronic or acute inflammation such as neurodegenerative diseases (parkinson's disease, alzheimer's disease and huntington's disease), atherosclerosis, certain forms of diabetes, autoimmune diseases, cancer, chronic wounds, damage caused by ischemia reperfusion, and Acute Respiratory Distress Syndrome (ARDS). It is well known that in the case of stroke, heart attack, grafts or ischemic wounds, etc., the metabolism can change significantly. As an example, mitochondrial respiration has been shown to play an important role in cardiac remodeling [ Kindo,2016], and cardiac metabolism responds to wall pressure via mitochondrial dysfunction [ Kindo,2012].

Therefore, strategies to normalize, restore and/or increase metabolism are of great interest for the treatment and characterization of many conditions. PBM therapy is one of these strategies [ hamlin 2017; hammblin 2018].

PBM therapy is based on light application at low (sub-thermal) irradiance, with a primary wavelength range between 600 and 900nm, and a spectral window corresponding to the maximum light penetration depth in most soft tissues. PBM has a broad range of molecular, cellular and tissue effects [ hamlin 2017; hammblin 2018].

However, the mechanism is not fully understood. In addition, PBM treatment parameters are rarely optimized and/or mastered. Studies based on several groups [ hamlin 2017; hamlin 2018], and most importantly, from in vitro and in vivo observations made by the inventors, it can be concluded that PBM produces several positive effects, in particular:

a) Tissue oxygen (O) after or during hypoxia ₂ ) The consumption is increased and the cost is increased,

b) The blood vessel formation is stimulated and the blood vessel,

c) The regeneration process is stimulated at the cellular level,

d) Increased endogenous production of protoporphyrin IX (PpIX) following application of 5-aminolevulinic acid (ALA) [ Sachar,2016 ]]Which can be used as O ₂ A sensor. It should be noted that several ALA formulations for eliciting PpIX are approved for cancer therapy and detection as photosensitizers and fluorescent markers, respectively.

e) An increase in ATP production, indicative of an improved metabolic activity,

f) Regulation of Reactive Oxygen Species (ROS).

g) Regulation of Reactive Nitrogen Species (RNS).

h) Rescue of toxic cells.

i) Embryo survival, affected by hypoxia/reoxygenation events, increases.

j) An increase in circulating Nitric Oxide (NO) during prolonged hypoxic or hypoxic events.

k) Continuous steady state during prolonged hypoxia or hypoxic blood events (based on hemodynamic variables, blood gas measurements as blood glucose).

These observations may originate from the stimulation of metabolic activity and are of great interest for numerous medical applications including those described above [ hamlin 2017; hammblin 2018].

PBM is of particular interest in the treatment of Myocardial Infarction (MI) [ Liebert,2017], which is one of the most common acute disorders. It is a major cause of death worldwide. Currently, the treatment options chosen for patients with MI to limit their size and reduce acute myocardial ischemia injury are time consuming, have side effects and have limited efficacy. They include direct percutaneous coronary intervention or thrombolytic therapy. Furthermore, the treatment itself (reperfusion process), which is also known as myocardial reperfusion injury, may be responsible for myocardial cell death until several days after treatment for which there has been no effective treatment until now [ choucani, 2016; ferrari,2017; kalogeris,2017].

PBMT is also of interest in the treatment of systemic inflammation when the situation is for fibromyalgia, arthritis associated with rheumatism or autoimmune diseases, especially when circulating blood is directly irradiated. PBMT may also help avoid the consequences of SARS-Cov2 during the acute phase, where strong immune responses generated by cell division storms may induce Acute Respiratory Distress Syndrome (ARDS), or during the chronic phase resulting from long-term SARS-Cov2 effects.

US2007/219604A1 discloses a method of applying PBM on a biological object, wherein light is delivered with a suitable temporal evolution of the optical power, which is determined based on the light delivery geometry on/in the biological object and the biological object optical coefficient. In this patent application US2007/219604A1, the PBM effect is caused by the generation of fluence rates in each part of the biological object volume in turn. It is noted that the application does not mention specific values of fluence rate and irradiation time.

However, existing methods for applying PBM are not efficient enough, in particular because of the bimodal effect of PBM, as described below.

The limited use of PBMT can also be explained by the lack of methods to monitor metabolic activity of biological tissues. This statement is supported by another finding by the inventors, which suggests that the importance of the PBM effect depends on the time at which the metabolically active light is applied relative to, for example, that determined by oxygen consumption.

There is therefore a need for improved use of PBM for treatment of biological subjects.

Drawings

Fig. 1: the fluence rate/irradiance pair for "broad", collimated and perpendicular illumination of air tissue interfaces versus semi-infinite tissue depth. The continuous curve is a solution of diffusion approximation, while the dashed curve is a solution based on Monte Carlo computer simulation, where μ _a Sum mu _s The absorption and scattering coefficients, respectively. Mu (mu) _eff Is the effective attenuation coefficient, g is the anisotropy factor, and k is the exponential front factor resulting from the back-scattering of light. Derived from [ Jacques,2010]。

Fig. 2: pO measured on single-layered HCM cells oscillated via metabolism ₂ (black curve; mmHg) and temperature (gray curve; DEG C).

Fig. 3: synergistic effect of STS and PBM on angiogenesis.

Fig. 4a, 4b: various PBM conditions, expressed as PBM/PBM-free PpIX fluorescence intensity ratio, which reflect in particular metabolic activity, were observed in Human Cardiomyocytes (HCM) at 689nm (fig. 4 a) and 652nm (fig. 4 b). The value of the ratio "PBM/no PBM" is given by the monochrome bar.

Fig. 4c: left: fluence rate dependent effects at 689nm (effective wavelength (potent wavelength)) and 730nm (ineffective wavelength (non-potent wavelength)) range from 0.5 to 15mW/cm ² ). Right: using an ineffective fluence rate (9 mW/cm ² ) A combination of an effective wavelength with an ineffective wavelength that has a significant effect on the relative increase in PpIX fluorescence.

Fig. 5: evolution of fluence rate/irradiance ratio (F/E) for "broad", collimated and perpendicular illumination of air tissue interfaces to semi-infinite tissue depth. The continuous curve is a solution of diffusion approximation, while the dashed curve is a solution based on Monte Carlo computer simulation, where μ _a Sum mu _s The absorption coefficient and the scattering coefficient, respectively. Mu (mu) _eff Is the effective attenuation coefficient, g is the anisotropy factor, and k depends on the index matching condition (n _tissue /n _air =1.37) and the optical coefficient, is the exponential front factor resulting from light back scattering. Derived from [ Jacques,2010]。

Fig. 6: pO in CAM at EDD 7 ₂ Is a frequency analysis of (a). Upper left: experimental images of a metallic clark probe applied against a blood vessel are shown. Left middle: pO (pO) ₂ Signals (acquired at 50 Hz). Left lower: the associated spectrum based on wavelet analysis (vertical scale is frequency and monochromatic level represents oscillation amplitude, horizontal scale is time in s). The right lower: pO (pO) ₂ Signals (acquired at 1 Hz) and top right: the monochrome level represents the oscillation amplitude of the associated spectrum produced by wavelet analysis. The horizontal axis is time given in minutes. pO was observed 25 minutes (out of scale time) after topical application of NaHS (10. Mu.l-1. Mu.M) ₂ Intense activation of tone, naHS, which was initiated by PBM (850 nm,7 mW.cm) ² 30 s) is deactivated (see myogenic signal). The horizontal line reported on the spectrum indicates a particular frequency. 1-heart, 2-breath, 3-myogenic, 4-neurogenic, 5-eNOS dept,6-eNOS index (possibly prostaglandin [ Shiogai et al)]). Heart frequency cannot be in samplingSolving the problem. There are no respiratory and nerve bands at this EDD.

Fig. 7: an enlarged view of the experiment is presented in fig. 6. The horizontal axis gives the time in minutes.

Fig. 8: left: images of in ovo hypoxia reoxygenation experiments. Right: pO recorded throughout the experiment ₂ Signal (OX-100 μm)) (vertical axis: mmHg). Due to N ₂ Flushing (up to 60 minutes) results in hypoxia followed by reoxygenation.

Fig. 9: stimulation of the chick embryo heart at EDD 5 by PBM during hypoxia cardiac arrest. Left: time course measurement of heart beat (assessed by changes in heart reflectivity in a region of interest defined by a rectangle presented on the image showing the embryo (right)). The heartbeat stops between 0 and 45 s. The heartbeat was then reactivated by PBM irradiation during 7s for more than 1min.

Fig. 10: spatial evolution of fluence rate (E) in semi-infinite tissue for "broad", collimated and perpendicular illumination of air tissue interfaces. Δz=0.3 mm; n=10; n is n _tissue/ n _air ＝1.37；

T＝180s；ΔF’＝1.6mW/cm ² The method comprises the steps of carrying out a first treatment on the surface of the The dashed curve corresponds to a continuous fit of the fluence rate "step-wise" evolution. The fitted analytical expression is presented as an inset in the graph. This means that E may be continuously changing rather than incrementally changing.

Fig. 11: time evolution of fluence rate (E) in semi-infinite tissue for "broad", collimated and perpendicular illumination of air tissue interfaces. Δz=0.3 mm; n=10; n is n _tissue /n _air ＝1.37；T＝180s；ΔF’＝1.6mW/cm ² The method comprises the steps of carrying out a first treatment on the surface of the The dashed curve corresponds to a continuous fit of the fluence rate "step-wise" evolution. The fitted analytical expression is presented as an inset in the graph. This means that E may be continuously changing rather than incrementally changing.

Fig. 12: overview of a 90 ° transapical implantation during coronary thrombosis (b) of a cylindrical dispenser (d) connected to a light source (a), the cylindrical dispenser passing through a maskPlaced across the myocardium in a predefined pattern and spacing. (e) An optical distributor,A catheter and a guide device to be inserted into the catheter for penetration. (f) The image shows the light transmission (652 nm-100 mW) from an isotropic distributor (sphere 0.8mm diameter) implanted in the center of the left ventricular myocardium of an ex vivo pig.

Fig. 13: in situ implantation following sternotomy, along the damaged left ventricle of the pig heartA catheter. A) The catheter is introduced from the apex side until it comes out B) from the top of the left ventricle). C) The heart cross section of the catheter was not removed after cardiac ablation. Where the catheter is placed well near the middle of the myocardial thickness.

Fig. 14a: visual localization of ischemic area (IZ) following left anterior coronary descending (LAD) occlusion of a partially perfused left ventricle during open heart surgery in pigs. IZ is easily distinguished from non-ischemic zones (NZ). Electrical impedance sensors may also be used to characterize ischemic areas.

AJP cardiac cycle physiology

Fig. 14b: after LAD occlusion during open heart surgery on pigs, a visual representation of the transapical light modulation described in example 1. During ischemia, the interstitial catheter is used for treating the ischemiaThe left myocardium is inserted from the apex to the left atrium, with the distance between them being optimal to maximize the treatment area. Then the cylindrical dispenser (+)>Rod length 7 cm) is placed at each +.>Is a kind of medium. PBM treatment is initiated for a few seconds to a few minutes depending on the irradiation time prior to reperfusion. Several seconds prior to ventricular reperfusion, used to670nm and 808nm from both dispensers. The catheter is then removed immediately after reperfusion. Finally, they can be placed in position after surgery for further regenerative irradiation.

Fig. 15: a simplified diagram shows a portion of an example of an apparatus for providing therapy. Depending on the disease and the method of treatment, e.g. interstitial or systemic, the device may be modular with a combination of different basic communication blocks. Some of the blocks may be based on observable sensors as described in this document, or interfaces to acquire data from common clinical systems. Some blocks may characterize the spectrum of illumination, while other blocks may be dedicated to controlling the exogenous perfusion and perfusion temperature into the catheter. Other actuators may also be added to the combined exogenous stimuli, such as a function of mechanical pressure or temperature of the PBM. In the case of a multi-lumen balloon catheter, as shown herein, some blocks may control the time and/or period and/or level of inflation/deflation, with or without regard to monitoring of changes. A portion of the device also integrates a specific balloon size and shape to optimally treat biological objects. For example, as shown, the balloon at the center of the right atrium assumes two opposite conical shapes, which optimally treat circulating objects from the superior and inferior vena cava. Furthermore, since PBM strongly affects biological rhythms, especially during hypoxia or hypoxemia, the balloon can be shaped to contact the sinoatrial node at the top of the right atrium or to contact the atrial-ventricular node. In addition, using a multilumen catheter, exogenous agents may be injected through the balloon either upstream or downstream of the irradiation function of the agent. Obviously, the device can also be used for photoactivation of photosensitizers. The blocks of these devices can also be implemented directly by various implants to minimize platelet aggregation and clotting on the heart valve. It may also be implemented, for example, on artificial heart or pancreatic chambers to increase biocompatibility/biostability, and/or may be implemented to reduce inflammation and immune response of implanted chambers used in chemotherapy, and as other examples, activate endothelialization of hip prostheses or vascular stents.

Fig. 16: examples of irradiance geometries used in PBM. Stippled areas schematically represent patterns of fluence rates in tissue. (a) (b) surface irradiation from a broad beam or lens head fiber. (c) - (e) irradiating with the cut ends or interstices of the cylindrical fibers. (f) - (h) intracavity and intracavity irradiation. (i) (j) intracavity full surface irradiation with isotropic tip fibres or light diffusing liquid (shadows).

The source is as follows: wilson,1986.

Fig. 17: spatial evolution of fluence rate (E) in semi-infinite tissue for "broad", collimated and perpendicular illumination of air tissue interfaces. The spatial evolution of E is optimized so that its value never exceeds 100mW/cm ² While minimizing the total irradiation time. The idea is to illuminate the sample with two PBM hot spots visible in fig. 4, i.e. generating 15 and 3mW/cm during 40 and 180s (T-value), respectively ² Is a function of the integrated flux rate of (a). The corresponding values of Δf', Δz and n (steps) are respectively:

-4mW/cm ² 0.15mm and 13 for t=40s

-1.6mW/cm ² 0.3mm and 4 last t=180 s.

n _tissue /n _air ＝1.37。

Fig. 18: time evolution of fluence rate (E) in semi-infinite tissue for "broad", collimated and perpendicular illumination of air tissue interfaces. The spatial evolution of E is optimized so that its value never exceeds 100mW/cm ² While minimizing the total irradiation time. The idea is to illuminate the sample with two PBM hot spots visible in fig. 4, i.e. generating 15 and 3mW/cm during 40 and 180s (T-value), respectively ² Is a function of the integrated flux rate of (a). The corresponding values of Δf', Δz and n (steps) are respectively:

-4mW/cm ² 0.15mm and 13 for t=40s

-1.6mW/cm ² 0.3mm and 4 last t=180 s.

n _tissue /n _air ＝1.37。

The "step-based" evolution of fluence rate can be fitted by an analytical expression presented as an inset in fig. 11.

Fig. 19a: illustrating non-uniformity along with non-uniform longitudinal emissivity from a cylindrical dispenserLight emissivity. The dispenser (2) is placed in a container delimited by a wall container (1). The emissivity of the dispenser is shaped to produce a light gradient along the rod (the light emitting portion of the dispenser) at all times, here represented by a fluence rate contour that is non-parallel to the rod. Considered at a certain speedBuilt-in at a certain distance h _i And h _j Two identical circulating objects passing all the way along the dispenser will be at specific positions a, respectively _i (h _i ) And A _j (h _j ) Find about by->Optimal fluence rate for defined irradiation time to address specific hot spots Ω _i,λ . Obviously, the optical gradient is adjusted according to the optical coefficient of the circulating medium, the geometry of the container, the speed level and the nature of the flow (laminar, turbulent, pulsatile).

Fig. 19b: the wavelength combinations of non-uniform emissivity are illustrated along with the continuous non-uniform longitudinal emissivity from the cylindrical dispenser. In some cases where a particular hotspot cannot be selected, for example, due to a mismatch between the constraint illumination time and the accessible fluence rate, an effective (λ) can be used _i ) And not effective (lambda) _j ) The combination of wavelength illumination bypasses this problem. In this scheme, in the case of any hot spot where any kind of cycle can be selected, the equal doses of the two wavelengths can be parallel, and then sequential or simultaneous uniform irradiation can be used to obtain an effective PBM effect. In contrast, if a hotspot of a certain cyclic object can be selected, non-uniform irradiation must be considered to optimally process all kinds of cyclic objects.

Fig. 19c: adjustment of the emissivity of the uniform longitudinal dispenser within the balloon. Will be(2 cm bars defined by radiolabels) are packaged into balloon-based catheters. The shape and size of the balloon is limited by the emissivity shape of the RDAnd (5) setting. In this case, the circulating objects passing near the balloon will be exposed to different fluence rates, as the distance within the dispenser is adjusted by adjusting the balloon diameter.

Fig. 20: cylindrical distributor for pig chest fluoroscopy display70 And) wherein the arrow positions the radiolabel defining the rod (7 cm). The rod starts from the superior vena cava, then passes through the right atrium, and ends in the inferior vena cava, as shown in fig. 15. The dispenser was placed following the procedure described in example 13, except that the catheter used was a peelable catheter that could be removed with the optical dispenser located only in the central venous line. The optical dispenser may allow for a duration of several days for long-term irradiation.

Fig. 21: irradiation planning schemes using hot spot "lines" at 689 nm. In FIG. 4, for a composition comprising 0.5 to 20mW.cm ^-2 A relatively efficient line appears at 40 s. This can be used to optimize irradiation treatment time by increasing the treatment depth per irradiation time. It is well known that fluence rates emanating from a gap longitudinal uniform distributor placed in a semi-infinite medium can be approximated by analytical expressions based on a second type of bessel function. Fig. 21 shows the optical coefficient (ua=0.17 mm ^-1 :ueff＝0.88mm ^-1 ) A treatment regimen of the evolution of the fluence rate defined internally perpendicular to the dispenser axis. Between 0 and 40s, 2.8mW.cm ^-1 Is coupled to the dispenser, which causes 20mW.cm near the surface of the dispenser ^-2 To a fluence rate of about 0.5mW.cm at-3.5 mm ^-2 Is a function of the integrated flux rate of (a). Between 40s and 80s, the optical power (multiplication factor P) was adjusted to 100mW.cm ^-1 So as to obtain 20mW.cm at 3.5mm ^-2 And the fluence rate at-7 mm reaches 0.5mW.cm ^-2 . Thus, using this hot spot line, in this case, the treatment depth was 7mm within a treatment time of 80 s.

Fig. 22: selection of a combined hot spot using the same wavelength at its particular illuminationA graphical representation of different portions of a biological subject being treated simultaneously over time. Based on the same simulation as described in fig. 21, in which a longitudinal dispenser is placed in the myocardium, the graph shows the evolution of the fluence rate of the continuous optical power applied over the depth of the tissue over the time indicated by the inset. Using hot spots Ω _i,689 ＝(20±1mW.cm ^-2 The method comprises the steps of carrying out a first treatment on the surface of the 60±1 s) with another hot spot Ω, for example, exhibiting a lower fluence rate but a multiple of the irradiation time _j,689 ＝(3±1.6mW.cm ^-2 The method comprises the steps of carrying out a first treatment on the surface of the 180±1 s) to treat the joint surface layer successfully every 60s (using Ω _i,689 ) While in parallel the second region of depth is accumulating therapy (the portion of fluence rate marked with black underline). During period a, three surface layers were treated (by every 60s relative to Ω _i,689 Increasing the optical power) (illumination time projected on the lower graph), whereas the depth area will receive a range of 3±1.6mw.cm ^-2 Is a continuous 3 x 60s. Since a portion of the depth region is subsequently received 180s, the optical power of this period is defined to avoid irradiating a portion that has been received 180 s. Then, as shown, the starting optical power of period B is defined as continuing Ω _j,689 This results in another part of the object being subjected to Ω _i,689 。

Fig. 20: cylindrical distributor for pig chest fluoroscopy display70 And) wherein the arrow positions the radiolabel defining the rod (7 cm). The rod starts from the superior vena cava, then passes through the right atrium, and ends in the inferior vena cava, as shown in fig. 15. The dispenser was placed following the procedure described in example 13, except that the catheter used was a peelable catheter that could be removed and the optical dispenser was located only in the central venous line. The optical dispenser may allow for a duration of several days for long-term irradiation.

Fig. 21: the illumination planning scheme illustrates how the illumination time can be minimized with a "long" cylindrical light distributor inserted into a "large" biological object using the "line" hot spot seen in fig. 4 a. In this geometry, the fluence rate varies with distance from the surface of the light distributor The evolution of (c) can be modeled by an analytical expression comprising a second class of bessel functions. For two different linear power densities expressed in mW/cm of the length of the optical splitter, the following optical coefficients (μ) for the biological object _a ＝0.17mm ^-1 :μ _eff ＝0.88mm ^-1 ) The evolution of the fluence rate as a function of distance as described above is shown. The first (2.8 mW/cm), using 40s, causes 20mW/cm at the light distributor surface ² And at a distance of 3.5mm, the fluence rate is about 0.5mW/cm ² . The second linear power density (100 mW/cm) was applied for between 40 and 80s, thus resulting in 20mW/cm at 3.5mm ² And at 7mm the value was 0.5mW/cm ² . Thus, the hot spot line was used for treatment for 80s with a treatment depth of 7mm.

Fig. 22: the use of two hot spots in combination (corresponding to surface Ω _i,689 And omega _j,689 ) To simultaneously treat illustrations of different depths of a biological object with one wavelength. Considering the geometric and optical conditions corresponding to fig. 21, the evolution of fluence rate with depth is shown for different linear power densities applied sequentially. Use of hotspots (Ω) _i,689 :20±1mW.cm ^-2 ；60±1s)、(Ω _j,689 :3±1.6mW.cm ^-2 The method comprises the steps of carrying out a first treatment on the surface of the 180±1 s), four layers were treated during two irradiation periods (a and B). During period a, hot spot Ω is used by increasing the linear power density by steps of 60s up to 180s _i,689 To treat two layers while the other two layers are using the hot spot omega _j,689 Treatment is performed at that time. As shown in the upper right hand corner of fig. 22, the total treatment time was 360s. It should be noted that the linear power density for period B is defined in such a way that the two treatment layers lying between 1 and about 3mm are continuous.

Fig. 23: this figure is a summary of figure 22 when applied simultaneously at two wavelengths exhibiting different penetration depths in tissue. Thus, the combined use of these two wavelengths can reduce the processing time mentioned in fig. 22 by a factor of 2.

Fig. 24a: the figure shows the normalized fluence rate around a tapered light distributor of length 7cm, expressed in (mW/cm) ² ) The letter/(mW/cm) indicates that the tapered light distributor is surrounded by a light guide having an optical property (. Mu.) corresponding to blood _a ＝0.25mm ^-1 ,μ _eff ＝1.07mm ^-1 ) Is a fluid of (a) a fluid of (b). The arrow indicates the blood volume element (blood volume element) propagating according to a trajectory parallel to the axis of the optical splitter, which trajectory is at a distance of 4mm from this axis. The figure illustrates that the blood volume element will be exposed to a desired normalized fluence rate independent of the location of the blood volume element.

Fig. 24b: the figure shows the same situation as in fig. 24a, but with the blood volume element propagating on the surface of the cone-shaped light distributor.

Fig. 25a: blood glucose quantification between the beginning and end of a hypoxemia event. PBM irradiation in deoxygenated blood during hypoxia significantly reduces blood glucose levels in blood.

Fig. 25b: during PBM irradiation in normoxic pulmonary arteries, the clark probe monitors arterial partial pressure in the aorta.

Detailed Description

The inventors have shown that the light dosimetry (fluence rate [ mW/cm) ² ]The method comprises the steps of carrying out a first treatment on the surface of the Light dose [ J/cm ] ² ]) And spectroscopy (wavelength) and control of the duration and time of irradiation are critical to cause optimal PBM effects. This observation is very important because it is known that the PBM effect is bimodal (sometimes referred to as biphasic), i.e. that fluence rates and/or light doses that are too high or too low significantly reduce the PBM effect, and therefore are often associated with the artdt-Schultz rule observed in pharmacology. Many groups studying various "standard" effects (mitochondrial membrane potential; ATP production; etc.) reported such bimodal responses [ Huang 2009; hamlin 2017; hamblin 2018]。

The effect of PBM on the endogenous production of PpIX in different cell lines, including glioma cells and Human Cardiomyocytes (HCM), was studied, and the inventors found that both fluence rate and irradiation time must be applied in a controlled manner. For a given illumination in each part of the biological object volume for optimizing the PBM effect, these two parameters have to be applied with specific values. Contrary to what is reported in the art, the inventors found that the bimodal effect of PBM was observed only for a specific set of these parameters. These parameter sets are defined in this document as "hot spots" (fig. 4a and 4 b). Furthermore, the inventors have shown that some of these hot spots are independent of wavelength.

It was also determined that the optical properties of biological tissue (described primarily by its absorption and scattering coefficients) have a significant impact on the propagation of light around the light source [ Tuchin,2015; hamlin 2017; hammblin 2018]. In general, the fluence rate (and light dose) decreases with distance from the light source due to absorption and scattering of light in the tissue (see fig. 1). Thus, in most cases, fluence rates and/or light doses in tissue will never be optimal simultaneously in different locations in tissue treated by PBM. The existence and the existence of the parameter hot spot are as follows: i) The combination of non-uniform distribution of light in tissue treated by PBM, and ii) very limited control of light delivery and dosimetry by most research or clinical teams active in this field, explains the limited and contradictory results reported in the literature [ Chung 2012]. This also explains why PBMT is currently poorly applied.

It is an object of the present invention to provide an improved PBM for use in the treatment of biological objects, such as tissues, circulating blood and/or lymph.

It is another object of the present invention to provide an efficient treatment of ischemia reperfusion injury, such as Myocardial Infarction (MI), by PBM applied in the above and below mentioned conditions and methods.

It is another object of the present invention to provide an efficient treatment of fibrillation, including atrial fibrillation, by the PBM applied in the conditions and methods mentioned above and below.

It is another object of the present invention to provide an efficient PBM-based treatment of metabolic disorders (e.g., type 2 diabetes, liver disease or hormone secretion) under the conditions and methods described above and below.

It is another object of the present invention to provide an efficient treatment of systemic inflammatory or exacerbated systemic immune response by PBM applied in the conditions and methods mentioned above and below.

It is another object of the present invention to provide an efficient PBM-based treatment to maintain systemic homeostasis during hypoxia and/or anoxia, under the conditions and methods described above and below.

It is another object of the present invention to provide efficient methods in cell-based therapies, particularly to increase proliferation rates of stem cells and trigger cell differentiation.

It is another object of the present invention to provide efficient PpIX-based treatment/diagnosis by fluorescence imaging PpIX, for example in photodynamic therapy or in cancer detection. Embodiments of the invention relate to: helmets using integrated light emitting diodes, which induce PBM irradiation through the skull over specific areas of the brain prior to photodynamic detection (PDD) or photodynamic therapy (PDT) procedures for controlling cancer.

It is another object of the invention to increase and homogenize the endogenous production of PpIX in plants and larvae. One example of such a method is to increase the efficacy of phototoxic effects induced in grasses/larvae.

It is another object of the present invention to provide for an efficient treatment of a condition by PBMT based on monitoring of metabolic activity. Such monitoring is based on frequency analysis of parameters reflecting metabolic activity, which enables to adjust radiometric parameters (fluence rate, irradiation time, light dose … …) and spectral (wavelength) parameters in such a way as to maximize the PBM effect. Such monitoring may also be used to evaluate the state of metabolic activity to determine the optimal light application moment. Embodiments of the present invention relate to the use of standard probes to measure biochemical parameters reflecting physiology of metabolic activity. As described below, such probes include thermocouples and Clark pO ₂ The probe or fiber-optic based probe measures these parameters. The signals delivered by these probes are then processed by a dedicated unit to perform frequency analysis, enabling extraction of parameters providing information about the PBM effects and metabolic activity.

The above object is achieved by the device and method of the invention as defined in the claims.

Advantageously, the device and method according to the invention is characterized in that the PBM effect is caused continuously in each part of the volume of the biological object by the generation of a specific fluence rate during a specific time corresponding to a specific "hot spot" as a selection condition (see below).

An illustrative embodiment of the present invention includes using one or more light sources coupled to one or more light distributors to apply a particular fluence rate during a particular time corresponding to one or more "hot spots" presented in fig. 4a and 4b to increase metabolism in the tissue/condition of interest referred to below.

The present invention also optionally includes an apparatus and method for predicting the time of application of PBM to a biological object based on frequency analysis of fluctuations in parameters reflecting metabolic activity (differential analysis of time signals (past integration or present derivative)) or based on artificial intelligence prediction methods reflecting one or more parameters of the PBM effect or metabolic activity of the biological object.

Optionally, the light power delivered by the device, the illumination time and the moment of change of the PBM application with respect to the metabolic activity are adjusted based on the feedback observables (see the list given below) to optimize the PBM effect.

Observable feedback:

a) Temperature.

Fig. 2 presents the evolution of the temperature measured with a thermocouple and a probe for measuring the Oxygen Consumption Rate (OCR) reflecting metabolic activity in an experimental setup developed by the inventors. The setup included a single layer of Human Cardiomyocytes (HCM) at the bottom of a petri dish covered with a 15mm thick layer of physiological water. Thermocouple and method for measuring OCR and partial pressure of oxygen (pO) ₂ ) Is located 1mm above the cell monolayer.

As can be seen from FIG. 2, when pO ₂ And therefore a significant and easily measurable change in temperature is observed when OCR is changed. The temperature increases with increasing OCR activity. Interestingly, in pO ₂ The OCR and high frequency oscillations of temperature observed after the maximum are in phase and synchronized.

Thus, measuring the temperature provides observable feedback to monitor and/or adjust the light dose for the PBM. Measuring the temperature also allows to determine the optimal PBM irradiation time with respect to metabolic changes.

b) Reflecting participationTissue in the redox state of the metabolized enzyme autofluoresces.

Oxidation is the primary process by which the necessary energy is generated in the cell. It may occur under aerobic or anaerobic conditions. In many cases, biological oxidation begins with substrate dehydrogenation, i.e., the replacement of two hydrogen atoms, while coenzymes such as nad+, nadp+ and FAD act as acceptors for these atoms. Because of the low intracellular concentration of these coenzymes, they must be recovered by reoxidation. Thus, these coenzymes act as the primary donor and acceptor in oxidative phosphorylation (OXPHOS) [ ferroesi, 2012 ] ]. Due to the binding of NADH and FAD to a number of enzymes involved in metabolic pathways [ Alberts,2002 ]]Thus, when the cell switches its metabolism, the relative ratio between NADH and FAD binding sites will also change [ Banerjee,1989 ]]. Thus, the cell pair produced by PBM can be monitored for O ₂ The response to changes in levels (changes in metabolic activity) was observed for their effects on FAD and NADH.

These coenzymes can be studied nondestructively and their autofluorescence observed, i.e. without the addition of exogenous probes [ Ramanujam,2001]. One of the most common optical techniques for providing information about the metabolic state of cells is based on determining the redox ratio of FAD and NADH by fluorescence spectroscopy (in particular time resolved fluorescence spectroscopy [ Skala,2007; skala,2010; kalina, 2016; walsh,2013 ]); walsh,2012; blackberry, 2016], a field corresponding to the expertise of the inventor for more than twenty years [ Wagni res,1998]. For example, in cancer cells, an increase in cellular metabolism is generally indicated by a decrease in redox ratio [ Chance,1989].

Thus, steady state and/or time resolved fluorescence spectroscopy (or imaging) of tissue autofluorescence is a interesting feedback observability for monitoring or adjusting the light dose for PBM. Interestingly, this approach was combined with exogenous pO based on molecular probes (PPIX or kalina et al ₂ Probe [ kalinia, 2016 ]]) Or direct O of time-resolved luminescence spectrum of interstitial Clark Probe ₂ Sensing is used in combination to provide unique information about the PBM effect. Monitoring these parameters is minimally invasive and rapid.

c) Blood vesselAssessment of hemoglobin saturation.

Under normal conditions, the body maintains a stable oxygen saturation level to a large extent through chemical processes of aerobic metabolism associated with respiration. However, it is well known that hemoglobin saturation varies due to different metabolic activities.

Since many methods are well established to measure hemoglobin saturation, particularly peripheral or central venous saturation, which is known to reflect cardiac output other than septic shock, it is of great interest to apply such methods to monitor changes in metabolic activity caused by PBM.

In addition, since various gases are endogenously produced and diffuse in tissues and circulating blood, and can bind to various kinds of metallic proteins exhibiting strong light absorption bands, for example, NO or H ₂ S can combine with deoxyhemoglobin to produce nitrosyl or sulfur or carboxin that reduces the level of available deoxyhemoglobin, and since PBM can cause photodissociation (photolysis) of the metallo-proteins to hemoglobin, especially nitrosyl [ Lohr,2009 ]Methemoglobin is formed simultaneously and changes due to these different forms of "hemoglobin" can be measured (by light absorption measurement [ Van leeuwen,2017]) It is therefore of great interest to monitor changes in metabolic activity caused by PBM by assessing these different metalloprotein complexes.

₃ ^- d) pH and/or bicarbonate (HCO) level.

Glycolysis is a well known metabolic pathway that converts glucose to pyruvate, which leads to acidification of the extracellular surrounding medium. This acidification is typically caused by lactate excretion following conversion of lactate from pyruvate [ Wu,2007].

Since many methods are well established to measure pH or bicarbonate levels in tissue or circulating blood, it is of great interest to apply such methods to monitor changes in metabolic activity caused by PBM.

Since tissue (or saliva) lactate concentration can be continuously assessed by a minimally invasive device such as a subcutaneous microneedle [ Tsurukoa,2016] or in the oral cavity, this method can be used to monitor changes in metabolic activity caused by PBM.

e) Concentration of ROS.

Reactive Oxygen Species (ROS) are chemically reactive oxygen-containing chemical species. Examples include peroxides, superoxides, hydroxyl radicals, singlet oxygen ([ Hayyan,2016 ]), and alpha-oxygen. In the biological context, ROS are formed as natural byproducts of normal oxygen metabolism and have an important role in cell signaling and homeostasis [ Devasagayam,2004]. ROS are produced during various biochemical reactions within cells and within organelles (e.g., mitochondria, peroxisomes, and endoplasmic reticulum).

The effects of ROS on cellular metabolism are well documented in various categories [ Nachiappan,2010]. These include not only roles in apoptosis (programmed cell death), but also positive roles such as the induction of host defense genes and the mobilization of ion transport systems. This implies that they control cellular functions. In particular, platelets involved in wound repair and blood homeostasis release ROS to recruit additional platelets to the site of injury. These also provide a link to the adaptive immune system by recruiting leukocytes.

Abnormal levels of ROS have been implicated in a number of pathologies through the strong regulation of various biological cascades [ Sies,2020]. Interestingly, ROS levels are for cell reprogramming [ Bigarella,2014; zhou,2016] and tissue remodeling are also native. For example, their kinetics of production depend on a broad spectrum of external or internal repetitive stimuli, such as hormone secretion or mechanical forces (e.g. vascular shear stress), which directly affect tissue behaviour and properties [ Hwang,2003; brandes,2014], and ultimately affect phenotypic aspects.

Since many methods are well established to measure ROS in tissues, it is of interest to apply such methods to monitor changes in metabolic activity caused by PBM.

Since ROS and Reactive Nitrogen Species (RNS) are intrinsically intricate [ moldazieva, 2018], and since many methods of assessing RNS are well established [ grocery, 2016], the term reactive oxygen species and reactive nitrogen RONS will be used herein. Indeed, in a broad sense, the term shall be active oxygen, active nitrogen and sulfur species (RONSS).

₂ f) Content of HS.

Hydrogen sulfide (H) ₂ S) exert a wide range of effects on the whole organism. It is an epigenetic regulator, in particular by DNA demethylation to cause histone modification, a process that allows cell differentiation [ Yang,2015 ]]. Which is indispensable in the aging process of aerobic organisms by maintaining high levels of mitochondrial DNA copy number [ Li,2015 ]]And activation by sirtuin 1 is essential in the aging process. Interestingly, H ₂ S is the only species that is both a substrate and an inhibitor of intra-mitochondrial OXPHOS based on its concentration [ Szabo,2014]This is probably consistent with the well-known observation that exogenous H ₂ S-inhalation causes a state of similar death in small mammals, called artificial hibernation or hypometabolism [ Blackstone,2005]. H is well known ₂ S prevents a number of heart diseases including heart failure caused by pressure overload [ Snijder,2015 ] ]. This supports the hypothesis that endogenous H ₂ S is a regulator of energy production in mammalian cells, in particular during stress conditions, which enables the cells to cope with energy demands in case of an oxygen deficiency [ Fu,2012 ]]。

Furthermore, we observed in ovo hypoxia reoxygenation in fertilized chicken eggs. This study was performed by combining H ₂ The S microprobe was gently placed over the ventricle or dorsal aorta of the chicken. We observed H ₂ The S level increases significantly during hypoxia (transient) arrest (or when the heart beats very slowly) and decreases upon re-beating. Thus, it appears that blood flow is through elimination of H ₂ Endogenous production of S plays a role, H ₂ S can bind deoxyhemoglobin to form and spread sulfur hemoglobin.

Since many methods can be well established to measure H in tissue ₂ S[Olson,2012]Thus, this method is applied to monitor generation caused by PBMChanges in metabolic activity are of great interest.

₂ g) Hydrogen selenide (HSe) levels.

Along with oxygen and sulfur, selenium (H) ₂ Se) belong to the chalcogen group and have similar excretion and metabolic pathways. Similar to H ₂ S，H ₂ Se is an endogenous small gas molecule that can cause pseudoid death states and shows reperfusion injury protection [ Iwata,2015 ]. Which reversibly binds to COX inhibiting mitochondrial respiration and is thought to bind to H ₂ S, NO and carbon monoxide (CO) together [ Kuganesan,2019 ]]. Furthermore, glutathione peroxidase is incorporated as a glutathione peroxidase in numerous selenoprotein oxidoreductases, which are critical for maintaining redox state homeostasis in health and disease, and its deficiency causes a substantial increase in ROS, which is suspected to be an important cause of cancer and CVD [ Bleys,2008]。

Since many methods are well established to measure H in tissue ₂ Se or selenium in serum, it is therefore of great interest to apply this method to monitor changes in metabolic activity caused by PBM.

h) Ion concentration.

Ions play an important role in the metabolism of all organisms, which is reflected in the various chemical reactions in which they are involved [ van Vliet,2001]. Ions are cofactors for enzymes, catalyzing basic functions such as electron transport, redox reactions, and energy metabolism; and they are also essential for maintaining the osmotic pressure of the cells. Ion homeostasis is critical for all living organisms, as ion confinement and ion overload both delay growth and can lead to cell death.

Since many methods are well established to measure ions (particularly calcium, potassium, chloride and/or hydrosulfide ions) in tissues, it is of great interest to apply such methods to monitor changes in metabolic activity caused by PBM.

i) Cytochrome levels, including cytochrome c oxidase, were monitored by NIRS.

It is well known that broadband near infrared spectroscopy (NIRS) can be used to monitor changes in concentration of cytochrome oxidation states, such as cytochrome-c-oxidase (Δoxcco), which plays a key role in mitochondrial respiration [ Roever,2017].

Since different methods are well established for measuring Δoxcco in tissues (or other cytochromes involved in metabolism), it is of interest to apply these methods (including NIRS) to monitor changes in metabolic activity caused by PBM.

j) Using medical (functional) imaging techniques (MRS "magnetic resonance spectroscopy"; MRI "magnetic resonance imaging"; NMR "Nuclear magnetic Co-ordination Vibrating "; PET "positron emission tomography"; EPR "electron paramagnetic resonance"; SPECT single photon emission computed tomography Drawing "; BOLD "blood oxygen level dependence"; NIRS "near infrared spectrum").

Metabolic imaging focuses on and targets changes in metabolic pathways to characterize various clinical conditions. Most molecular imaging techniques are based on PET and MRS, including conventional techniques in thermal equilibrium ¹ H and ¹³ cMRS and hyperpolarized magnetic resonance imaging (HP MRI). Di Gialleonado et al review metabolic pathways altered in many pathological conditions and corresponding probes and techniques for studying these alterations [ Di Gialleonado, 2016 ]]. Furthermore, fuss et al [ Fuss,2016 ]]The use of medical imaging to address various conditions in humans is described.

Since many metabolic imaging-based methods are well established to assess metabolism, it is of great interest to apply these methods (including functional metabolic imaging) to monitor changes in metabolic activity caused by PBM.

k) Vascular tone and vasomotor.

Vascular tone refers to the degree of constriction undergone by a blood vessel relative to its maximum expanded state. All arterial and venous vessels under basal conditions have a degree of smooth muscle contraction between the balance of the contracting and expanding muscle effects, which determines the diameter of the vessel, e.g. vascular resistance to adapt/regulate blood flow and pressure. Basal vascular tone varies between the large circulation, the microcirculation and the organs. Certain organs have greater vasodilatory capacity (e.g., myocardium, skeletal muscle, skin, visceral circulation) and therefore high vascular tone, while other organs have relatively lower vasodilatory capacity (e.g., brain and renal circulation) and therefore low vascular tone.

The degree of vascular tone is regulated differently between macroscopic (arteries, veins) and microscopic (arterioles, venules, capillaries). Notably, even though tone may be regulated by extrinsic factors (nerves, circulatory metabolites), blood vessels exhibit spontaneous oscillations (vasomotor), causing blood flow movements [ Aalkaejer,2011]. Thus, analysis of vascular tone through dependence of the tone in various actuators from local to systemic can provide insight into metabolic activity and can reflect the degree of aging [ Bentov,2015] and many pathophysiological conditions such as ulcer risk, type 2 diabetes [ Smirnova,2013], endothelial dysfunction or hypertension [ Ticcinelli,2017], renal disease [ Loutzenhiser,2002] [ Carlstrom,2015] or metabolic syndrome [ Walther,2015]. Furthermore, assessment of the endothelial function of the cutaneous microvasculature was used as a diagnostic and prognostic symptom of CVD [ Hellman,2015].

Since many methods are well established to assess vascular tone and vasoconstriction, such as video capillary microscopy, plethysmography [ Tamura,2019], laser doppler flow meters, pressure measurements such as by Cutaneous Vascular Conduction (CVC) or time-frequency analysis, and since from a dynamic point of view all the cardiovascular system is considered to be a single entity coupled to an oscillator [ Shiogai,2010], any method capable of assessing changes in metabolic activity caused by PBM, including Heart Rate Variability (HRV) giving information about the autonomic nervous system by ECG or by Phonocardiogram (PCG) [ heart sound measurement of Alvarez, 2014], is of great interest 2018. As observed by the inventors during surgery, respiratory frequency variability (RFI) [ stevanovska,2007] or ballistocardiography is of great interest to monitor changes in metabolic activity caused by PBM.

l) electromagnetic endogenous signalIs used.

Electrocardiography (ECG), electroencephalogram (EEG) and Electromyography (EMG) are standard measurements of electrical activity of heart, brain and muscle metabolism, respectively. Novel ECG analysis based on signal computation classification [ Patidar,2015] is expected to be a tool for cardiac diagnosis, in particular, it is possible to understand in depth coronary artery diseases [ Kumar,2017], [ Acharia,2017], arrhythmias and ischemic diseases [ Bhoi,2017]. The same type of analysis was performed on EEG, showing interesting results of seizures (the most common brain disease) [ Bhattacharyya,2018].

Since many methods are well established to monitor these electromagnetic endogenous signals, methods that are capable of assessing changes in metabolic activity caused by PBM are of great interest.

m) use of bioimpedance signals.

It can be used clinically to measure various physiological parameters [ Petterson,2016]. This method is used for cardiac pacemakers such as Ensite from St Jude Medical, optiVol from Medtronic, and closed-loop stimulation from biotronik.

Since many methods are well established to measure bioelectrical impedance in tissue or directly on skin, any of these methods that are capable of assessing changes in metabolic activity caused by PBM are of great interest.

n) the presence of a marker in the circulating blood.

Long lists of circulating markers of interest that monitor light dose during PBM include metabolites (succinate, pyruvate, etc.), clotting factors, apoptosis factors, (pro-and anti-) inflammatory factors, as well as liver factors, mitochondrial cytokines (mitokins), or isolated mitochondrial levels, for example. It should be noted that only a few of these are listed here.

Blood glucose level: many conditions are associated with deregulation of circulating glucose levels, which can directly lead to systemic metabolic disorders, as in the case of diabetes. Therefore, monitoring changes in metabolic activity by assessing blood glucose is of great interest.

Succinate: succinate is a key intermediate of the tricarboxylic acid cycle (TCA) cycle, playing an important role in anabolic and catabolic pathways. Furthermore, it is significantly associated with reperfusion injury [ choucani 2014]. Mitochondria are a physiological source of succinic acid, but accumulated succinic acid may be transported into the cytosol and then into the circulating blood. This TCA cycle intermediate links the intracellular metabolic state and the intercellular signal, which is denoted [ Tretter,2016]. Succinate levels in blood vary from 2 to 20 μm, where the concentration increases with hypoxia stress, pro-inflammatory stimuli, exercise or pathological conditions such as type 2 diabetes, obesity or ischemia reperfusion injury [ grimizzi, 2018]. Since circulating levels of succinic acid can be monitored by bioluminescence assays or raman spectroscopy, it is of great interest to evaluate changes in metabolic activity caused by PBM by circulating levels of succinic acid.

Lactate and Lactate Dehydrogenase (LDH): LDH is a common marker of cell damage and cell death. In addition, PBM can reduce LDH generated during anaerobic exercises [ Park,2017]. Therefore, it is of great interest to use these markers to assess changes in metabolic activity caused by PBM. Furthermore, the combination of LDH levels with aspartate Aminotransferase (AST) levels can be an effective indicator of body tissue damage. It should be noted that the lactic acid level can be estimated directly on saliva using a new air-metering method [ Tamura,2018].

Due to high levels of metabolic activity in inflammation or immune response, in particular the ability to alter its phenotype by immune cells, serum/plasma levels of immune/inflammatory markers, such as: mtDNA copies, leukocyte count, total antioxidant capacity, bicarbonate, malondialdehyde (MDA), uric acid, bilirubin; cytokine or chemokine marker levels, for example, such as interleukin IL2, IL6, IL7, IL10, IL18 or tnfα, and macrophage inflammatory protein 1- α, IP10, MCP1; and because lymphocyte T and/or monocyte M2 activation by flow cytometry is also of great interest.

Serum/plasma levels of thioredoxin: the level of this enzyme is elevated in infections, ischemia reperfusion, and other oxidative stresses. They are therefore good markers for monitoring oxidative stress. Thioredoxin plasma levels are also elevated in coronary spasmodic angina and other cardiovascular disease patients [ Nakamura,2004].

Cardiac markers: several established markers (myoglobin, creatine kinase isozymes, troponin I and T, B type natriuretic peptides, transaminases) are used clinically for diagnosing myocardial infarction and also for diagnosing other organ injuries. To a lesser extent, LDH, glycogen phosphorylase and recently ischemia modified albumin can be used for diagnosis in 30 minute assays [ Dasgupta,2014]. This is also the case for thioredoxin levels [ Jekell,2004].

Levels of circulating eNOS and NO or nitrite or nitrate: the levels of these compounds are important, especially for regulating systemic blood pressure and systemic homeostasis [ Wood,2013]. To begin with, the loop H can also be evaluated ₂ S or sulfite or sulfate levels to monitor changes in metabolic activity caused by PBM.

Circulating mitochondria: recently it has been shown that cell free functional mitochondria are present in circulating blood. Furthermore, mitochondrial cytokines (mitokynes) are important in metabolic remodeling, especially in heart failure [ Duan,2019]. Therefore, monitoring changes in metabolic activity by assessing circulating mitochondrial levels or mitochondrial cytokine levels is of great interest.

o) optical mapping of cardiac electrical signals using voltage sensitive fluorescent dyes.

Although optical imaging of cardiac electrical signals using voltage sensitive fluorescent dyes (VSFs) has only been performed in experimental studies, since these VSFs have not been approved for clinical use, FDA approved dyes, such as indocyanine green (ICG) [ Martisiene,2016], exhibit voltage sensitivity in various tissues, thereby increasing the hope that electrical activity of cardiac tissue can be mapped clinically. Therefore, methods based on mapping (or locally evaluating using a "point measurement" system) cardiac electrical signals using voltage sensitive dyes to monitor/adjust the light dose during PBM are of great interest.

p) assessing various tissue oxygen using redox sensorsAnd a state of the reduced state.Since metabolism and redox reactions are intricate and since many methods evaluate metabolic activity based on measurements of redox sensor proteins, monitoring changes in metabolic activity caused by PBM with redox indicator probes is of great interest.

q) assessing the status of various tissues, including cardiac tissue, using ultrasound.

Ultrasonography is a well established method of examining heart tissue. Many parameters characterizing heart tissue and blood flow are typically obtained during an ultrasound examination.

Therefore, methods based on the use of ultrasound inspection to monitor the light dose during PBM are of great interest.

_{2 2 2} r) reflecting each using pO (and/or OCR) by measuring arterial partial pressure of oxygen (PaO) and partial pressure of inhaled oxygen (FiO) Metabolic activity of the tissue and/or the whole body.

pO ₂ Measurements can be readily made in exogenous or endogenous probes of different compartments (tissues or organs) or of different organelles within a cell. For example, such probes may be detected optically. Other techniques, such as EPR oximeter, polarographic electrodes or BOLD imaging, are of great interest for assessing changes in metabolic activity caused by PBM.

s) use of hemodynamic variables.

In the clinic, these variables must be evaluated in real time to monitor metabolic activity, especially in the case of cardiovascular injury. Such measurements relate mainly to arterial and venous gas pressure, cardiac output, stroke volume, capillary vessel pressure and systemic and pulmonary resistance. Therefore, it is of great interest to use these methods to assess changes in metabolic activity caused by PBM.

t) use of the Krebs cycle enzyme kinetics.

It is well known that the krebs cycle enzyme kinetics are good markers of metabolism, particularly for assessing the levels of mitochondrial proteins. For example, since the activity of a Gu Dingmei or succinate dehydrogenase is generally measured clinically, it is of great interest to use these methods to assess changes in metabolic activity caused by PBM.

w) PpIX level

Protoporphyrin IX is a precursor to many organometallic proteins, such as hemoglobin and chlorophyll. The inventors have shown that, PBM treated cells tend to increase their endogenous production of PpIX. Therefore, it is of great interest to use methods based on detecting PpIX levels to assess changes in metabolic activity caused by PBM. Since heme concentration is a feedback parameter in the PpIX endogenous production pathway, it is of great interest to extend to measure circulating hematocrit levels to assess changes in metabolic activity caused by PBM.

x) monitoring metabonomics and lipidomics, in particular oxyipine (oxyipine).

Oxylipidins are biologically active metabolites derived from the oxidation of polyunsaturated fatty acids. Furthermore, they play a key role in cardiovascular thrombosis and risk factor progression. Therefore, their monitoring is of great interest.

y) glycoprotein levels

Glycoproteins, including proteins and carbohydrate chains, are known to be involved in many physiological functions, including immunity. They possess a receptor signaling domain that recruits signaling molecules.

z) glycerol level

Glycerol can be used as a marker of apoptosis. One of the functions of glycerol is to act as a chemical partner. In particular, it has the ability to enhance expression of apoptosis-regulating factors (bax).

aa) the assessment of the immunomodulatory effects caused by PBM can be monitored by pro-inflammatory circulating monocytes such as CD14, CD16, which can differentiate into dendritic cells. It can also be assessed by cytokine profile of macrophages.

ab) monitoring the basis of oxytocin levels. In the latter case, it has been shown to monitor oxytocin levels in the intensive care unit of premature infants as a relevant marker of pain.

Optionally, the optical power, duration of irradiation and application time of the PBM defined by the device are combined with the application of an exogenous stimulus, wherein the stimulus may be an agent that increases the effect of the PBM (see the list given below). It should be noted that the time between the administration of the exogenous agent and the PBM irradiation may take into account the duration of assimilation as well as the kinetics of activation of the agent.

The inventors have demonstrated that the combined administration of PBM with, in particular, an exogenous agent (sulfur donor) and a nitric oxide donor results in Synergistic effect.

As the inventors have shown, co-administration of ALA with PBM increases the accumulation of PpIX and thus increases the level of endogenous PpIX. Thus, co-administration of ALA and light is of great interest for increasing the PBM effect. It should be noted that other exogenous agents may be combined in the PBM to increase its effect, as shown below.

As shown in fig. 3, the inventors have recently observed that angiogenesis observed on chick embryo chorioallantoic membrane (CAM) is even more stimulated when PBM is used in combination with FDA approved exogenous, sodium Thiosulfate (STS) (sulfur donor). This strongly suggests that the combination of PBM with exogenous such agents stimulates angiogenesis or metabolic activity more than PBM alone or such agents.

The assessment of these PBM effects on angiogenesis was performed using a method based on fluorescence angiography performed on CAM several days after PBM. FIG. 3 presents a general CAM fluorescence angiography (left panel), which was quantitatively characterized using image processing and analysis software developed by our laboratory [ Nowak-Sliwinska,2010]. The main objective of this development was to characterize the dynamic changes that occur in the capillary/vascular network of CAM between day 6 and day 12 embryo development (EDD), monitoring CAM vessels using an epifluorescence microscope equipped with a scientific camera after intravenous (iv) injection of fluorescent agent [ Nowak-Sliwinska,2010]. From the generated angiogram, 3 descriptors are extracted: per mm ² Branch point number, average area of the vascular network grid, and average value of the third quartile of the grid area histogram. As shown in fig. 3, we The proof of concept results demonstrate that PBM significantly stimulates CAM angiogenesis. This effect is even more pronounced if PBM is combined with exogenous application of 175mM STS.

Interestingly, at the same time, the inventors monitored H on chick embryos ₂ S and NO levels are observed in ovo, and topical application of STS causes a significant increase in NO over a long period of time (6 to 12 hours), whereas when PBM irradiation is performed 1-2 hours after STS application, the time to generate NO is significantly reduced, typically to one hour.

Since STS is clinically approved H ₂ S donor [ Snijder,2015]It is used to prevent a number of heart conditions, as for H ₂ S has been reported as [ Yu,2014 ]]Including heart failure by pressure overload caused by upregulation of endothelial Nitric Oxide (NO) synthase [ Kondo,2013]Renal ischemia/reperfusion injury [ Bos,2009 ]]Use of PBM in combination with H administration in conjunction with the above devices/protocols ₂ S donors (e.g. STS or methylsulfonylmethane (MSM), or e.g. dithiothione, or other compounds exhibiting different H' S ₂ S kinetic released donors) and/or NO donor substances, such as arginine, including NO itself, are of great interest.

Since cysteine is an important source of sulfides in human metabolism, the co-administration of such protein amino acids or derivatives thereof, e.g. selenocysteine, or synthetic forms such as N-acetylcysteine, is of great interest for enhancing the effect of PBM.

MSM is a naturally occurring organic sulfur compound that is used as an alternative source of bioactive sulfur. It is mainly used for anti-inflammatory treatment. It has been studied in animal models and in many human clinical trials [ Butawan,2017]. The antioxidant capacity of MSM is also recognized, and it has been proposed that the antioxidant mechanism acts indirectly through mitochondria, rather than directly at the chemical level [ Beilke,1987]. MSM is well tolerated by most people at doses up to 4 grams per day as an FDA approved substance with few side effects [ Butawan,2017]. Results of in vivo and in vitro studies indicate that MSM is responsible for oxidative stress and inflammatory cross-talk at the transcriptional and subcellular level [ Butawan,2017]. Interestingly, kim et alHuman ([ Kim, 2009)]) MSM has also been shown to reduce inducible expression of Nitric Oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) by inhibiting nuclear factor- κB (NF- κB), a transcription factor involved in immune and cellular stress responses. This observation is very interesting because NO is a powerful vasodilator that is involved in many metabolic functions. With other gas conveyers (known as gas conveyers) [ Donald,2016 ] ]) As such, NO will have a specific effect on its local concentration and microenvironment [ Thomas,2015 ]]This may affect many different processes [ Rapozzi,2013; reeves,2009]. PBM has also been proposed to cause NO photodissociation from COX [ Karu,2005; lane,2006]. At the same time, NO photodissociation from other intracellular "reservoirs" (e.g., nitrosylated forms of myoglobin and hemoglobin) is also assumed [ Lohr,2009]. It has been established that NO production by mitochondrial NO synthase down regulates cellular respiration. Replacement of O by NO from COX ₂ Can inhibit cellular respiration and ATP production [ Antunes,2004; cooper,2008]. Thus, PBM is believed to increase ATP production. Alternative and possibly parallel mechanisms explaining the release of PBM and/or increasing the bioavailability of NO may be related to the role of COX as nitrite reductase (single electron reduction of nitrite to NO), especially when O ₂ Low partial pressure [ Ball,2011 ]]。

Taken together, these observations suggest that MSM has an indirect effect on the mitochondrial Electron Transport Chain (ETC) through its NO regulation. Furthermore, analysis of the literature shows that PBM is associated with NO donors (e.g.S-nitrosothiolsOr alkyl nitrite, including NO itself) causes a strong synergistic effect.

In addition, due to H ₂ The interaction between S and NO can produce nitroxyl (HNO), which plays an effective role in oxidative stress and cardioprotection in the cardiovascular system, systole, angiotensin, and angiogenesis [ Nagpure,2016; wu 2018]Thus, co-administration of (H) NO with a nitroxyl donor, cimlanod or hexyl 1-nitrosocycloacetate is of great interest for increasing the PBM effect.

Ebselen is an FDA approved H ₂ Se donors, which are interesting for increasing the PBM effect, as the inventors have discussed (page 13Point g).

As already mentioned, NAD ⁺ Is essential for redox reactions and it controls hundreds of key processes of energy metabolism for cell survival, rising and falling according to food intake, exercise and time of day. Thus, as vitamin B3, NAD in PBM ⁺ Administration of the donor is of great interest for increasing the PBM effect.

Other exogenous agents of interest for use in combination with PBM are:

curcumin, which is the main active ingredient of turmeric (l.), is known to have a variety of effects on both healthy and cancerous tissues. Notably, curcumin causes ER stress, resulting in unfolded protein responses, primary retrograde signaling, and calcium release, which disrupts the stability of mitochondrial compartments and causes apoptosis.

Dexmedetomidine, a well-known α2 agonist for anesthesia, has recently attracted interest because it is believed that dexmedetomidine pretreatment reduces myocardial ischemia/reperfusion injury by inhibiting mast cell degranulation. Similarly, EPO has shown a positive effect in lipopolysaccharide in ischemia reperfusion injury and arterial vascular injury in the kidney.

Ivermectin, approved by the FDA for the treatment of humans suffering from intestinal roundworm-like and onchocerciasis, both diseases caused by parasites. Furthermore, clinical evidence supports that the use of ivermectin reduces mortality in patients infected with SARS-CoV 2. The combination of ivermectin and PBM may reduce the dose of ivermectin, in particular reducing side effects.

Vigore as a source of nitrite in various forms (e.g. alkyl nitrite)

By extension, any exogenous agent known to regulate metabolism, for example, particularly by regulating ETC or ROS modulators to regulate metabolism in mitochondria, is of great interest to increase PBM effects. This is the case for Adenosine Diphosphate (ADP) which is known to increase OCR or for vitamin K, ketamine succinylcholine, acetylcholine and atropine, bradykinin. Other agents include catecholamines such as epinephrine, norepinephrine, or dopamine, opioids or various kinase modulators that activate various G proteins or various antioxidants and/or anti-inflammatory donors, such as resveratrol. Finally, targets for rapamycin or deacetylase modulators are of great interest to increase PBM effects.

To extend, due to temperature, exogenous or endogenous mechanical stress [ Li,2005; hwang 2003]Physical exercise as well as electrical stimulation, hyperoxia, hemostasis (remote pretreatment) are known to regulate metabolism, any exogenous stimulus or combination of environmental/physical/electrical or electromagnetic stimuli as applied to biological subjects are of great interest to increase PBM effects. For example, endogenous H is well known ₂ The level of S is inversely related to temperature. The increase in temperature in situ can be considered as an indirect endogenous H ₂ S donor, and conversely, an increase in situ temperature can be considered endogenous H ₂ S inhibitors.

As observed by the inventors, the efficacy of PBM is not only dependent on the light dose [ J/m ] ² ]And spectroscopy (wavelength), but surprisingly also on the fluence rate for a particular illumination time. For example, the inventors observed that the specific case shown in FIGS. 4a and 4b, a 3mW/cm period of 3 minutes was necessary ² Irradiance (i.e. dose of 0.54J/cm) ² ) The cells were irradiated (equal to the fluence rate in this particular environment consisting of cell cultures). The different irradiation times and/or irradiance do not cause any PBM effect, except for e.g. at 15mW/cm ² By 40s or 25mW/cm during 22 seconds ² "hot spots" observed under irradiance of (c). Interestingly, some of these hot spots are independent of wavelength, as a comparison of fig. 4a and 4b can conclude that the hot spots are present at the same location in terms of irradiance and illumination time.

This is a more complex illustration of the PBM response, i.e. the fluence rate and/or light dose being too high or too low, significantly reduces the PBM effect compared to the well known PBM bimodal effect. The inventors have also demonstrated that under certain conditions, there is no "neutralization" of the PBM effect by the excess dose/irradiation before and/or after application of the PBM at optimal conditions.

When in useThe inventors have also observed surprising results when the PBM is performed with a combination of wavelengths, wherein one of the wavelengths is ineffective when used alone. This is the case for 730nm, which wavelength is not effective when used alone, as shown in fig. 4 c. As can be seen from the figure, the wavelength of 689nm is 3mW/cm ² Is effective for 180s of irradiation time at 9mW/cm ² Is ineffective for irradiance applications. Surprisingly, 730nm (irradiance of 3mW/cm ² For 180 s) and 689nm (irradiance of 9mW/cm ² The simultaneous or sequential combination of irradiation for 180 s) produced a pronounced PBM effect, as evidenced by PpIX fluorescence intensity ratio (PBM/no PBM). These effects are comparable to the effects corresponding to the "hot spot" shown in fig. 4a and 4 b. Thus, a simultaneous or sequential combination of wavelengths in such a way that at least one of them is inactive (or poorly active) is of great interest to increase the PBM effect, especially when specific hot spots of active wavelengths are difficult to reach due to optical or geometrical limitations.

In fact, the optical properties of biological tissue (mainly by its absorption coefficient μ _a And a lower scattering coefficient mu _s ' described) has a significant effect on the propagation of light around the optical splitter, which is well established. In general, fluence rates (and light doses) decrease with distance from the light source due to absorption and scattering of light in the tissue for a given power (and illumination time). FIG. 5 illustrates that the optical element is in the "wide" (much larger than μ) _eff ^-1 ) In the specific case of a semi-infinite sample of collimated illumination, how the fluence rate F (normalized by irradiance E) decreases with distance from the surface. Thus, fluence rates and light doses in tissue are never optimal at the same time at different locations of tissue treated by the PBM.

It should be noted that the geometry of the light distributor (illumination geometry) is adapted to the specific organ to be treated. For example, front side (wide area), balloon-based or interstitial irradiation using one or more optical fibers (see the commercial product of Medlight SA "http:// www.medlight.com/#", as an illustrative example) is specifically contemplated.

The inventors also established that the metabolic activity is based on reflectionIn particular using wavelet theory) to adjust the radiometry and innovation of spectral conditions used in PBMT. More precisely, they have an oxygen partial pressure (pO) of chick embryo chorioallantoic membrane (CAM) during PBM ₂ ) Time-frequency analysis was performed.

It is well known that arterioles, in particular in the peripheral microcirculation, are regulated by complex metabolism [ Reglin,2014]pO to surrounding tissue ₂ Reaction is intense [ Jackson,2016 ]]In which there is a low frequency oscillation of blood perfusion [ Kvandal,2006]。

Based on the use of commercially available Clark probesOX-needle, OX 100-Fast (Fast)) for pO in CAM during "long" (several hours) time ₂ The local measurements performed, we calculated the slave pO ₂ Is a frequency spectrum obtained by wavelet-based analysis. Wavelet analysis is a well known mathematical transformation that is capable of characterizing non-stationary frequencies during the measurement time. FIG. 6 (left middle) shows pO near the new CAM arterioles at embryonic day (EDD) 7 ₂ Is a typical measurement of (a). Here, the time signal (measurement time: -180 s) is mainly generated by superposition of the following 2 frequencies (see fig. 6, bottom left; vertical axis is frequency): heartbeat (-1 Hz) and myogenic tension (-0, 1 Hz) that represents the intrinsic activity of vascular smooth muscle.

H ₂ S is an effective regulator of vascular tone [2012]It can be caused by administration of NaSH. We measured the H of CAM arterioles induced by topical application of NaSH (10. Mu.l, 1. Mu.M in physiological serum) using our Clark probe ₂ S stimulation produced pO around 60mmHg ₂ Strongly regulated. This modulation was observed at least for myogenic (0, 05Hz-0,15 Hz), endothelial nitric oxide synthase related frequency band (0.01 Hz-0.02 Hz) and endothelial nitric oxide synthase unrelated frequency band (0.005 Hz-0.01 Hz). Other lower bands are also activated, wherein this is clearly indicative of some of them being associated with prostaglandins or precursors from endothelial cellsProstacyclin release is associated.

Our innovation stems from our use of front side light distributors (850 nm,7mw cm ^-2 30 s) of PBM illumination performed on the CAM (see fig. 7). The adjustment ("dimming") of these frequencies (fig. 6 (upper right) and fig. 7) is obviously caused by the PBM.

In particular, bands 3, 5 and 6, and bands corresponding to undefined lower frequencies, appear to be totally or partially suppressed by the PBM (see fig. 6). Inhibition of band 3 (myogenic band) suggests that since this band is due to arteriole smooth muscle cells, it is caused by activation of NADPH Oxidase (NOX) and subsequent ROS production [ Nowicki,2001; li,2017]PBM inhibits NOX activity. Since the NOX superfamily plays an important role in inflammation and immune responses, where NOX in particular is involved in the metabolic turnover of leukocyte activation, our observations reveal that PBM regulates an important pathway of inflammation and is effective as an important immunomodulator. Furthermore, modulation of the myogenic tension in a biological subject by PBM can prevent many hypertensive lesions, as is the case in renal lesions [ Loutzenhiser,2002; moss,2016 ]. In summary, our results indicate that the parameters reflecting metabolic activity (e.g., pO ₂ ) Can monitor the light dose and/or fluence rate for the PBM. It should be noted that for illustrative purposes, i.e., increasing the signal-to-noise ratio, the oscillations observed in FIGS. 6 and 7 are those observed by H ₂ S. In fact, even in the absence of H ₂ This oscillation also exists in the case of S.

Therefore, it is of great interest to adjust the radiometric and spectroscopic conditions used in PBM therapy based on frequency analysis of parameters reflecting metabolic activity.

This particular type of monitoring may be performed for two main purposes: i) The PBM light is applied at an optimal time with respect to metabolic "oscillations", or ii) the level of metabolic change caused by PBM is evaluated in a way that is optimal (to adapt to radiometry).

As just presented above, pO ₂ Not the only parameter that is analyzed using wavelets or based on frequency to monitor the light dosimetry during PBM. Upper rowThe list (feedback observables list) describes other parameters of interest:

the inventors have also shown that the application time of light irradiation within a biological object is critical to cause significant PBM effects. These observations are important because biological objects are dynamic over a wide frequency scale of metabolic activities triggered by transient or conventional endogenous or exogenous factors. Notably, the inventors have shown that when light is applied at a specific time during the metabolic activity of glioma cells or HCM, the metabolic response of the cells is significantly differently modulated, thereby modulating the phenotype long-term response accordingly. The inventors also showed that using the in ovo chick embryo heart model during hypoxia/reoxygenation studies, when PBM irradiation was started just before reoxygenation, survival was significantly higher compared to when PBM was performed during long periods of ischemia before reoxygenation or after reoxygenation. Interestingly, under this condition, it was observed that reoxygenation caused a cardiac arrest during a time ranging from one second to several minutes. PBM modulation prior to reoxygenation significantly avoided this cardiac arrest. Thus, the inventors have shown that PBM restarts or regulates heart beat after hypoxic cardiac arrest or bradycardia or tachycardia occurs without observing the effects on a healthy beating heart. These in ovo observations have been confirmed by the inventors in vivo during an ischemic/reperfusion event caused by ligation of the coronary arteries of the pig heart.

The inventors also demonstrate that PBM can be used to modulate heart chicken embryos during hypoxia reoxygenation events.

One aspect of the invention is the use of the device or method to treat ischemia reperfusion injury, particularly those affecting the heart muscle, to reduce infarct size following an acute Myocardial Infarction (MI).

Based on chicken embryo hearts, the inventors developed an in ovo hypoxia reoxygenation experiment in which eggs were placed in a constant temperature air chamber in which the environment and embryo temperature and pO were continuously monitored ₂ . For some experiments, small H ₂ S, NO and pH probes are also positioned at different locations around the embryo heart or embryo tissue. The chamber was placed under a microscope for image recording. After a stabilization time (temperature stabilization), an anoxic environment is formed without any change in the ambient temperature by flushing nitrogen gas around the eggs during several tens of minutes, and the eggs are subsequently reoxygenated, as shown in fig. 8.

At Embryonic Development Day (EDD) 3, pure N was flushed during 45 minutes prior to embryo reoxygenation ₂ Resulting in a mortality rate of more than 50% 48 hours after the end of the experiment. This experiment supports one aspect of the "reperfusion injury" mentioned as "oxygen paradox" in Latham et al (Latham, 1951), i.e., reperfusion may be fatal in some cases (Piper, 2000). In our case, and for isolated chicken heart embryos (Raddatz, 2010), reoxygenation causes bursts of Reactive Oxygen Species (ROS) and permanent or transient cardiac arrest, followed by irregular heartbeats (bradycardia, tachycardia). In our experiments, we observed that the embryo was photomodulated just prior to reperfusion when the embryo at EDD 3 underwent 45 minutes of hypoxia (671 or 806 nm,5mw. Cm ² 30 s) significantly avoided cardiac arrest and importantly increased 48 hour survival. Interestingly, if the light is applied too early or too late after reoxygenation, no such positive effect of PBM is observed. The last observation clearly shows that the time of application of light relative to reoxygenation is critical for producing beneficial results.

Thus, one application of great interest to the present invention includes the use of PBM delivered by our original medical devices and methods to treat lesions resulting from hypoxic reoxygenation events and lesions that are extended to ischemia reperfusion events.

The inventors have also shown that PBM can stimulate heartbeats after hypoxia non-permanent cardiac arrest in the chick embryo heart.

Oxygen supply to chicken embryos occurs primarily by diffusion across the shell and then through the embryo prior to Embryonic Development Day (EDD) 7. The heart beat and blood flow that can be observed from EDD 2 are mainly the stimulus for cardiovascular development. Embryos until day 5 flatten on a "surface" located directly under the albumin layer. Thus, it is easily accessible after removal of a portion of the shell. This is why, consistent with chicken embryogenesis, embryos ranging from EDD 2 to EDD 5 have been used as excellent models of developmental biology for decades, particularly in terms of cardiac and cardiac rhythm generation. The model was also used for hypoxia-reoxygenation studies [ Sedmera,2002], where their behavior was studied during and after hypoxia or hypoxia, but also during hypoxia-induced tachycardia, bradycardia or for fibrillation studies.

One interesting PBM effect observed by the inventors in-ovo hypoxia experiments is related to the positive effect of light, which enables restarting the heart after cardiac arrest. In fact, prolonged hypoxia can lead to a cardiac arrest, and the heart sometimes restarts beating briefly until an irreversible complete cardiac arrest occurs. The inventors observed that in most cases, consistent with resuscitation observations on ischemic pigs, PBM irradiation frequently restarted beating hearts following such cardiac arrest (fig. 9). Furthermore, based on frequency and phase shift analysis of the subchambers of the embryo heart (atrium, ventricle, outflow tract) beating, PBM irradiation stimulates the recovery of this frequency (especially harmonics) and the phase shift of the contraction between subchambers after hypoxia. This is of particular interest because this phase shift must be preserved to maintain the efficacy of the pump function (cardiac output). Thus, by monitoring the frequency and phase shift of the motion of the heart sub-chambers, as shown by the inventors, it is known that localized illumination of sub-chambers (particularly the atrium) is very sensitive to hypoxia and is of great interest in maintaining cardiac output.

This surprisingly positive effect of PBM strongly suggests that it triggers metabolic activity involving the heart beat, including after hypoxic cardiac arrest. PBM is of great interest, for example, in the treatment of conditions such as tremors (including atrial fibrillation) because it is known that PBM can reduce inflammation and promote metabolic activity.

By extension, since metabolism is subject to both autonomous (i.e. cell cycle independent [ Papaginankis, 2017 ]) and non-autonomous rhythms, as is the case for example with circadian rhythms [ Bailey,2014], the use of PBM light at specific times and/or frequencies to lock in, trigger and/or (re) synchronize metabolic oscillations is of great interest, especially for the treatment of various metabolic disorders, such as type 2 diabetes [ Petrenko,2020], metabolic conversion into aerobic glycolysis (Warbugg effect) in cancer cells [ Gatenby,2018] or liver disorders and diseases [ Zhong,2018 ].

The inventors have also shown that delivery of PBM light directly in the blood perfused large vessels (porcine pulmonary artery, vena cava) or in the right atrium containing deoxygenated blood can be used to modulate systemic hemodynamics and oxygen tension, resulting in anti-inflammatory, immunomodulating, anti-aggregation, endothelial cell and epithelial cell protection. Surprisingly, based on gas measurements performed in arterial and venous blood (using cobas b 123POC System Roche) This irradiation of deoxygenated blood maintains homeostasis during the long-term hypoxic event of the pig. In addition, these exposures maintain and stabilize functional hemodynamic variables, such as cardiac output, accompanied by heparinized NO probes (NO-NP) placed in the pulmonary artery or atrium, e.g., during the period of several ten minutes after PBM exposure ) An increase and stabilization of the systemic unsteady NO level measured internally. This effect is unexpected because it is generally believed that the lifetime of labile NO in blood is ten to hundred times shorter. Interestingly, there was no concomitant increase in the assessment of methemoglobin in venous or arterial gas during the experiment. Furthermore, the inventors have shown that PBM light delivered directly in the blood perfused large vessels (pulmonary arteries) or the right atrium containing deoxygenated blood can be used to control hypoxia, hemoglobin saturation, arterial and venous oxygen partial pressure associated with hypoxia. Furthermore, this approach can be used to maintain blood glucose levels (fig. 25 a) in order to significantly reduce the likelihood of causing systemic tissue damage, as is the case with Multiple Organ Failure (MOF) caused by blood glucose disorders.

Detailed description of the invention:

according to the results in FIGS. 4a and 4b showing the PBM effect on the ability of HCM cells to produce PpIX, 3mW/cm must be applied during 3 minutes ² Is (or is strong)Degree) to maximize this effect. Since fluence rates are not uniform in volumetric (or 3D) tissue for a fixed irradiance, as shown in fig. 5, for a particular geometry and a particular optical coefficient, a medical device according to the invention preferably delivers irradiance that varies over time in such a way that all cells receive the optimal fluence rate during 3 minutes. In most cases, the illumination geometry will not change during the illumination process. Thus, irradiance is simply determined by the power of the irradiated tissue [ W ]Surface divided by the irradiation point [ m ] ² ]Given. Although Yarosplavsky et al [ reference ]]A similar concept has been described, but one finding of the inventors mentioned herein is to determine specific values of fluence rate and illumination time that must be applied simultaneously.

Let us consider the specific case corresponding to the geometry and optical coefficient mentioned in fig. 5. Since the fluence rate F can be determined by a solution to the diffusion approximation (note: the assumption that the basis at the diffusion approximation must be satisfied, i.e.: i) μ _a <<μs'; and ii) we are studying "far" from the light source and boundary (i.e. z>1/μ _s ') fluence rate at position "z"; "k" is a factor generated by light backscattered by the tissue to increase fluence rate at the interface, e.g. Jacques [ Jacques,2010]Described).

F＝k.E.e ^{-μeff z} Equation 1)

The only way to keep F constant (hereinafter F') to increase the value of z is to increase E. The statement results from the inversion of the last expression, written as:

E＝(F’/k).e ^{μeff z} equation 2

Since each HCM cell must be operated at F' =3 mW/cm during 3 minutes ² Another important concept is related to the tolerance affecting the fluence rate. If it is necessary to use a power of exactly 3mW/cm ² Irradiating cells, the total treatment of the whole sample will take an infinitely long time, since the volume corresponding to these cells is equal to 0mm ³ (they are confined to a plane at depth "z", which has a depth equal to 0 mm) ³ Is the volume of (c). However, looking at FIG. 4a showsAt 3mW/cm ² And an irradiance full width at half maximum (FWHM) of about 3.2mW/cm for a "peak" at 180s ² I.e. fluence rate F' must be 3.+ -. 1.6mW/cm ² In the range (hereinafter denoted as F '±Δf') to cause the optimal PBM effect after 180 s. This means that the HCM cells in which the best PBM effect is induced are not in the plane, but in the slice with a thickness Δz. The thickness Δz of the slice is given by:

because E= (F'/k). E ^{μeff z} We obtain:

Δz＝ΔF’/μ _eff f' equation 4

Therefore, Δz depends only on F ', ΔF' and μ _eff 。

If the volume of tissue to be treated is in the range z ₁ (proximal position) and z ₂ (distal position), then the number "n" of different irradiance applied during 180s (hereinafter T) is equal to: z ₂ -z ₁ /Δz。

Thus, the spatial evolution of irradiance E is shown in fig. 10.

The temporal evolution of irradiance E (t) is (see fig. 11):

E(t)＝(F’/k).e ^{μeff.Δz.t/T} ＝(F’/k).e ^{ΔF’.t/F’.T} equation 5

Wherein when 0 is<t<At T, E will be equal to (assuming the diffusion approximation equation is valid) F'/k.e ^ΔF’/2F’ When T<t<At 2T, equal to F'/k.e ^{3ΔF’/2F’} When 2T<t<At 3T, equal to F'/k.e ^{5ΔF’/2F’} When nT<t<(n+1) T, equal to F'/k.e ^{(2n+1)ΔF’/ 2} F', where n=μ _eff .F’.(z ₂ –z ₁ ) Δf' (see equation 6 below).

Thus, the treatment range is z ₁ And z ₂ The total time "t" required for tissue volume in between _tot The method is as follows:

T.(z ₂ -z ₁ ) /deltaz. By explicit expression of Δz (equation 4), we get:

t _tot ＝T(z ₂ –z ₁ )/(ΔF’/μ _eff .F’)＝T.μ _eff .F’.(z ₂ –z ₁ ) Delta F' equation 6

Finally, it should be noted that, like F', T can be applied with a certain tolerance due to the FWHM of the PBM peak along the irradiation time axis (see fig. 4 a).

In summary, in this example, the device according to the invention is in the range 0 and t _tot The irradiance E (t) is applied during the time in between, given by: e (t) = (F'/k) ·e ^{ΔF’.t/F’.T} 。

Since the device is powered by a specific power PW]Specific area of illuminated surface sm ² ]We therefore get E (t) =p (t)/S.

Thus, the device is in the range of 0 and t _tot The optical power P (t) is delivered during the time in between, given by:

P(t)＝(S.F’/k).e ^{ΔF’.t/F’.T} equation 7

E (t) and t _tot All parameters involved in the expression are for (known thickness z ₂ -z ₁ Determined by the specific organ and the irradiation geometry (of the surface S): in practice, F ', ΔF' and T are derived from FIG. 4a (for HCM), while k is derived from the tissue optical coefficient (known for the tissue of interest).

This detailed description of the best PBM effect for solving the entire volume of a particular hot spot of fig. 4a and 4b may be summarized as "hot spot line" or "hot spot surface" Ω _iλ Where i represents a particular hot spot surface on the graph presented in fig. 4d at wavelength λ. Since the points presented in fig. 4a and 4b represent specific coordinates (fluence rate; illumination time), and since the points around the maximum of the hot spot may have reasonable efficacy, the hot spot may be described as being represented by a rectangle Ω _iλ (ΔF’ _iλ ；ΔT _iλ ) Defined area: hot spot surface Ω _iλ Is typically equal to omega in size _iλ Average value of points in the interiorOr center of gravity, as shown in fig. 4 d. Can be defined to correspond to a specific omega _iλ (which has a specific dimension DeltaF' _iλ And DeltaT _iλ ) In order to minimize the total treatment time within the concomitant reduction of the expected PBM effect, as described in more detail below. The minimization of cost algorithm can then define the optimal hot spot (spot, line or plane) based on user-defined parameter definitions, based on the treatment case (acute, chronic, severe, moderate) and the geometry of the treatment area, in particular by defining the maximum treatment time value and the expected minimum treatment efficacy level. For example, for the treatment of superficial wounds, it is optimal to use the point of hot spots, wherein in this case the treatment time is not too long due to the smaller wound depth. In the case of acute infarctions, however, the time is urgent and the treatment volume is also of considerable importance, the hot spot spots can be converted into Δf' _iλ And/or DeltaT _iλ The face of the relatively high hot spot is treated in the shortest time to treat as much total volume as possible. It should be noted that most hot spots are located at 0.2J.cm ^-2 To 1J.cm ^-2 But other hot spots may be present in the irradiation time range of the order of seconds, the corresponding fluence rate is per cm ^-2 Hundreds of mW.

In the description given above, the tissue is considered stationary, while the power of the light source varies over time to produce an optimal fluence rate in the target tissue during an optimal time. However, there are certain situations, for example in fluids comprising blood, where the geometry is dynamic, for example due to blood flow. In such cases, the power delivered by the light distributor may be stable (without time evolution), but the light pattern produced by the light distributor, combined with the fluid optical properties, may allow the fluence rate to be optimized in certain capacity elements due to the fluid flow. Therefore, it is necessary to introduce longitudinal variations in emissivity in such a way that the light dose and/or fluence rate is optimal in order to induce PBM effects at different locations of the moving fluid (e.g. blood). An illustrative example is presented in fig. 19a, where a non-uniform longitudinal light distributor is located in the center of a blood vessel. Due to the emissivity (W/cm of the optical distributor ² ) Increase of the irradiation window along the sameAdditionally, a blood volume element located close to the surface of the dispenser will thus obtain the proper irradiance on the left side of the image, while a blood volume element located away from the surface will obtain the proper irradiance on the right side of the image. Thus, the whole blood volume will receive the optimal dosimetry as it moves along the cylindrical light distributor.

Based on the surprising results obtained by the inventors, which demonstrate that the effect resulting from the use of PBM effective wavelengths applied under suboptimal radiometric conditions can be optimized by the combined application of non-effective wavelengths, another PBMT scheme can be defined. An illustrative example (fig. 19 b) involves PBMT irradiation, where both the active and inactive wavelengths are delivered continuously. Wavelengths may also be delivered at the same location of the dispenser. The optimal optical longitudinal distribution profile depends on many parameters including vessel diameter (geometry), blood flow, its state (laminar, turbulent, pulsatile aspects … …) and blood optical properties. The device can be easily manufactured with minor changes to the process used to achieve a uniform cylindrical light distributor, or by designing a specific balloon catheter shape/size, as shown in fig. 19 c. The use of the balloon (inflation, irradiation, deflation) may be static or dynamic, just like the counterpulsation balloon used in the aorta, inflated or deflated during weeks in synchronization with the heartbeat. The time of flight (time of the subject around the rod) can also be adjusted to optimize PBM treatment. This may be done, for example, based on the regulation of cardiac output (using NG-monomethyl-L-arginine (L-NMMA), or by the inflation level or inflation/deflation cadence of a balloon-based catheter around the light distributor.

Example 1: treatment of myocardial ischemia-reperfusion

A detailed example of an apparatus according to the invention is presented in this example.

Such treatment of myocardial ischemia-reperfusion may be performed during:

1. during acute or chronic Myocardial Infarction (MI).

2. Aortic occlusion and cardiac arrest following extracorporeal circulation.

3. During organ transplantation (heart, lung or otherwise).

The light distribution route considered is:

1. interstitial (warp heart muscle)

2. Cavity (indoor)

3. Intravascular (intracoronary)

Medical device for warp and heart muscle

This involves implantation by the cardiac surgeon through the heart, after the acute phase of MI or during revascularization of the myocardium after aortic occlusion for more than 120 minutes under extracorporeal circulation or during heart transplantation, of a light distributor, preferably cylindrical and based on one or more optical fibers (fig. 12, 13 and 14 b).

These light distributors are placed in the diseased heart area (e.g. ischemic area) at the end of the surgery before or during coronary reperfusion.

These light distributors are placed according to the procedure described below:

1) The treatment region is located and estimated by:

a. macroscopic assessment of affected area (FIG. 14 a) (visual indicators: edema, akinesia, dyskinesia, vermilion appearance and/or use of characterization instruments)

b. Correlation with coronary angiography and ultrasound data corresponding to the following ischemic myocardial anatomical region (e.g., left ventricle):

i. front region (Anterior territory)

Side region (Lateral territory)

Lower region (Inferior territory)

2) Determining the number of light distributors required for light delivery and their relative positions based on the extent and accessibility of the ischemic area:

a. in situ consideration of the distribution of the coronary arteries to avoid their penetrative fixation (trans-fixation)

b. The light distributors are fixed and implanted penetrating at an angle between horizontal and surface normal, in the thickness of the myocardial layer (according to ultrasound data in time-shifted mode), to maximize the volume to be treated by the distributors and to allow them to be fixed as close as possible to the epicardium

c. The length of the light distributors (5 cm) is larger than their maximum length in the myocardium to distribute the light over the whole myocardial thickness.

3) A semi-rigid and transparent silicone-type or biodegradable mask is placed and fixed over the epicardial surface of the heart to be treated through 4 points (single strands of 5.0 wire). The mask can be used as both a guide/template for piercing the fixation (pre-drilled at the correct implantation angle and its holes follow a geometric pattern and predefined spacing that allows for the propagation of light in the tissue depending on the wavelength of light used). The mask also serves to hold the puncture fixation catheter into which the light distributor is inserted.

4) Using, for exampleThe catheter is used for fixing the myocardial wall through mask puncture, < + >>The characteristics of the catheter are:

a. less than 20 gauge (to minimize bleeding and tissue damage),

b. the hollow part of the hollow part is provided with a hollow,

c. transparent at the wavelength of use and,

d. a closed end cap, a sealing cover and a sealing cover,

e. visual indicia on the catheter indicates the length of catheter placement.

5) Connected to an optical splitter device and then subjected to an optical calibration step

6) Introducing a light distributor into each conduit (similar toInterstitial processes used in photodynamic therapy). The dispenser is attached to the catheter by means of a "luer lock". All light distributors are attached to the mask and/or the patient skin.

7) The temporal evolution of the light power emitted by the light distributor is determined such that the treated cells receive the best spatial irradiance ("fluence rate") in the best time. The determination is made based on the number and location of the light distributors determined by the user. For example, it consists in deriving the temporal evolution of the optical power emitted by the optical splitter from a preset weighting matrix. These matrices are generated based on monte carlo-type simulations of light propagation in the tissue of interest and a cost reduction algorithm that takes into account the specificity (optical and biological) of each wavelength for optimizing the processing time.

The optical delivery is performed in such a way that the temporal evolution of the optical power of the light source is performed according to the determination described in the previous step. Which can be adjusted by monitoring methods based on various instruments or biological data as previously described. A simplified diagram is presented in fig. 15, illustrating an example of a portion of an apparatus for providing an optical splitter.

In the case of a single irradiation:

i. surgical reperfusion procedure

Catheter and mask removal

Hemostasis, if necessary at the puncture fixation point

Verification of light distributor calibration

And v, continuing the surgical intervention as usual.

Normal closing procedure as any sternotomy.

In the case of multiple irradiation:

i. surgical reperfusion procedure

The catheter and light distributor are placed in position and attached to the biodegradable mask. The light distributor may be passed through the cannula into the skin, fixed and placed in place for 8 to 10 days in order to remotely repeat the process.

Surgical intervention is continued as usual.

Normal closing procedure as any sternotomy.

Removal of the light distributor is performed with the chest drainage in place by simple manual removal accompanied by ultrasonography at the second hour.

Intra-luminal medical device

This involves percutaneous placement of one or more light distributors into the left ventricle under fluoroscopy by an interventional cardiologist during myocardial revascularization in the acute phase of MI, in a pre-treatment, treatment or post-treatment.

These light distributors are placed at the beginning of the procedure and then add blood supply to the occluded coronary heart vessels.

These optical splitters are implanted according to the following procedure:

1) Access to the radial or femoral artery is achieved by ultrasonic puncture.

2) A radiation focusing (radio) guide sheath (5F-6F) was introduced using the Seldinger method.

3) Under fluoroscopy, the catheter is navigated to the left ventricle using radiation focused diagnostic guidance after crossing the aortic valve. The guide is removed and placed through a diagnostic catheter with or without a self-expanding systolic balloon in the left ventricle, or a light distributor fixed directly to the ischemic left ventricle wall.

4) Light delivery. The determination of the temporal evolution of the optical power emitted by the optical splitter and the optical delivery are performed as described above.

5) Removal of the catheter, light distributor and sheath; the device may be left in place for 8 to 10 days.

6) The conventional procedure of reperfusion of an occluded coronary artery by interventional radiology.

Intravascular medical device

This involves the percutaneous placement of one or more light distributors in an artery during MI, lung or other organ transplantation that suffers from ischemia reperfusion phenomena, during pre-treatment, treatment or post-treatment, under fluoroscopy, during revascularization following ischemia phenomena in interventional radiology, whatever the artery is.

These light distributors are placed at the beginning or end of the process before reperfusion.

These optical splitters, in the case of coronary arteries, are implanted according to the following procedure:

1) Use of I.V. catheterRadial or femoral artery movement)The pulses are close.

2) Radiation focusing sheath (5F-6F) was introduced using the Seldinger method.

3) Under fluoroscopy, navigation is performed using a diagnostic catheter (4 f, jr 4) and a 0.035 "guide (radiation focusing) to the coronary arteries.

4) The guide and placement of the light distributor is removed through the diagnostic catheter.

5) Light delivery. The determination of the temporal evolution of the optical power emitted by the optical splitter and the optical delivery are performed as described above.

6) Removal of the catheter, light distributor and sheath.

7) Coronary reperfusion procedure as usual.

These processes can be transferred to heart transplantation because it is well known that ischemia reperfusion injury is a major problem during organ transplantation, which is a major cause of graft rejection.

By extension, the above procedure can also be applied to other organs that suffer from ischemia reperfusion injury, as is the case, for example, for kidneys, liver, spleen or brain. These processes may be combined with other processes to simultaneously irradiate different parts of the body, such as the thyroid gland, to control possible negative systemic reactions caused by the organ suffering from I/R.

Example 2: continuous time variation of irradiance (or power) emitted by the light source.

Since Δf' defined in the detailed description is greater than zero, the temporal evolution of irradiance (or power) delivered by the light source may be continuous rather than incremental (as shown by the dashed line fitting the histogram in fig. 10 and 11). In this case, the temporal evolution of the irradiance or power delivered by the light distributor is given by equations 5 and 7, respectively.

Example 3: the irradiance (or power) emitted by the light source decreases over time rather than increases.

Different tissue layers of thickness deltaz may be irradiated with an appropriate fluence rate while increasing or decreasing (incrementally and/or continuously) irradiance (or power).

In this case t _tot The same, but time evolution of irradiance (or power) is given by the following expression, respectively: e (t) = (F'/k) ·e ^{ΔF’.(ttot-t)/F’.T} Or P (t) = (S.F'/k) e ^{ΔF’.(ttot-t)/F’.T} ，(0<t<t _tot )。

Example 4: different illumination (light delivery) geometries.

Fig. 5 shows the spatial distribution of fluence rates for a particular geometry (i.e. in semi-infinite tissue illuminated with broad, collimated and perpendicular beams at the air-tissue interface). It is well known in the field of photomedicine, in particular in photo-bioregulation or LLLT, to consider different illumination geometries depending on tissue/organ structure and pathway. Fig. 6 illustrates some of the most common illumination geometries.

There are solutions to the diffusion approximation for many of these geometries to determine fluence rates. Thus, for other illumination, organ, and/or light delivery geometries, the general expression of equation 5 can be written as: e (t) =f _E (μ _a ,μ _s ,g,n _ext ,n _tissue F ', ΔF', S, T, T), where F _E Is a function that depends on tissue optical parameters, organ and illumination geometry, fluence rate and its FWHM and illumination time that produces local maxima of the PBM effect. Many different methods are known for determining F _E As described in example 6 below.

Similarly, the general expression of equation 7 for other illumination, organ, and/or light delivery geometries can be written as: p (t) =f _P (μ _a ,μ _s ,g,n _ext ,n _tissue F ', ΔF', S, T, T), where F _P Is a function that depends on tissue optical parameters, organ and illumination geometry, fluence rate and its FWHM and illumination time that produces local maxima of the PBM effect. Many different methods are known for determining F _P As described in example 6 below.

A combination of the light delivery geometries presented in fig. 16 may also be used. Obviously, it is certainly also conceivable to use light sources in a direct or quasi-contact manner, such as ex-situ or in-situ LEDs or VCSELs.

Finally, it is certainly also possible to envisage heterogeneous organization, in particular hierarchical organization.

Example 5: different tissue optical properties.

The above form is specific to mu due to the different optical properties of different types of tissue _a 、μ _s 、g、n _ext 、n _tissue Is effective, especially if these optical properties change for a given tissue during irradiation.

Example 6: the fluence rate is estimated using other methods than diffusion-based approximation.

Different approaches have been established to model the propagation of light in biological tissues [ marteli, 2009]. Thus, these methods may be used to determine the temporal evolution of the light source to produce the optimal PBM effect instead of or in combination with the form of diffusion approximation based on the light transmission equation described above. These methods are developed mainly in the field of photo-medicine, and are used to master the dosimetry of light in tissues, and are divided into two categories:

1) The analysis method comprises the following steps: in addition to the well-known diffusion approximation of light transmission theory (which is used to establish the forms set forth in the detailed description of the invention above), other analytical methods are well established in the art, including but not limited to: the Kubelka-Munk theory, delta-Eddington radiation transmission equation.

2) A computer-based method of: many computer-based methods have been proposed for decades to simulate the propagation of light in biological tissues. These methods include, but are not limited to: monte carlo simulation, finite element simulation.

Example 7: different tissue responses to PBM are considered depending on cell type, wavelength (spectral design) and metabolic activity.

F' (3 mW/cm) ² )、ΔF’(1.6mW/cm ² ) And T (180 s) from our samples (human cardiomyocytes: HCM), environment (medium, temperature, pO ₂ Etc.) and spectral design (only at 689nmNext, one irradiation) was performed. However, in particular, changing one or a combination of these conditions may result in different values of F ', Δf' and T. This is especially the case if the time diagram of the application of the light changes.

Thus, the concepts presented above can be generalized to different conditions and cell types.

Example 8: in radiometric conditions (irradiance/fluence) corresponding to a plurality of "hot spots" in fig. 4a and 4b Rate, duration/dose) of irradiation of tissue.

It should be emphasized that the number of the components,FIGS. 4a and 4bSeveral "hot spots" are presented. Two "hot spots", i.e. the one mentioned above, corresponding to 3mW/cm ² Irradiance and irradiation time of 180s (hot spot 1), and irradiance and irradiation time of the second are 15mW/cm, respectively ² And 40s (hot spot 2).

This is important, in particular, to minimize the total treatment time.

In practice, since a "high" irradiance cannot be applied without damaging the tissue, only tissue located "close" to the illuminated surface can be irradiated with 15mW/cm ² Is treated with a "high" irradiance. Otherwise, cells close to the light source may experience thermal damage when cells at a distance receive a relatively high fluence rate.

It is widely accepted in the scientific community in this field that if the cross-sectional area is wide (diameter greater than μ _eff ^-1 ) Is applied with hundreds of mW/cm during a period of time exceeding a few seconds ² The thermal effect will begin to be noticeable if the irradiance of red (or NIR) light.

FIGS. 17 and 18 below illustrate that if irradiance must be less than 100mW/cm ² (other conditions that are the same as those considered in fig. 10 and 11), how the presence of "hot spot 2" is exploited to minimize processing time.

In this case, the treatment algorithm is as follows:

so long as E<100mW/cm ² The temporal evolution of the irradiance (or power) is then calculated by equation 5 #Or 7) wherein: f' =15 mW/cm ² ；ΔF’＝4mW/cm ² And t=40s. Otherwise, the value of F' must be used = 3mW/cm ² ；ΔF’＝1.6mW/cm ² And t=180 s.

This example can be extended by using a combination of wavelengths within their own hotspots.

Example 9: using passive attenuators to generate irradiance (or work) using a Continuous Wave (CW) light source according to equations 5 and 7 Rate) of the time evolution of the rate.

There are many commercially available light sources for treating tissue by PBM. Needless to say, none of them produces irradiance (or power) according to equation 5 (or 7). However, since many of these commercial light sources emit CW light and generate more than 0.62mW/cm ² (under our specific conditions, 3mW/cm ² Divided by k=4.87), they can be combined with attenuators so that they change their transmission over time in such a way that the irradiance corresponds to the value given in equation 5.

More precisely, if E' is the irradiance generated at 689nm by such a source without an attenuator, the attenuator transmits (T _r ) The time evolution of (2) will be given by: t (T) _r (t) =e (t)/E', where E (t) will be given by equation 5.

In summary, the specific design of the device according to the invention must combine a CW light source with one or more attenuators to finally obtain irradiance corresponding to that given in equation 5.

The generalizations mentioned in examples 1 to 8 above also apply to this example.

Example 10: high frequency adjustment of light superimposed on the temporal evolution of irradiance or power given in example 4 is used.

Because biological objects have dynamic optical absorption and response to light, due in part to their dynamic change in redox state, the wavelength or multiplexing wavelength for PBM can be synchronized/tuned at higher frequencies than the time variation defined in equation 5, taking into account the dynamics of oxidative metabolic redox states.

Example 11: according to equations 5 and 7, irradiance (or work) is modified by a pulsed light source using pulse duration variations Rate).

As already mentioned in example 10, the light may be adjusted at a higher frequency than the frequency for irradiance (or power or fluence rate) variation according to equations 5 and 7. Since the average power P (t) is the time average of the following pulsed optical power P (t):

P(t)＝∫p(t)dt

the time evolution of P (t) can be varied for a given frequency and peak power by adjusting the duty cycle of P (t).

Example 12: causing bystanders or remote effects.

As observed by the inventors under total blood volume irradiation performed in the central venous line paO ₂ And other arterial gases (e.g., chloride ions) occur only in arterial blood, either transiently or intermediately but with significant regulation. In fact, these modulations were not observed in the central venous blood (see figures 25b and c). Since PBM is known to cause bystander or remote effects under certain conditions, it is of great interest to irradiate different parts of a biological object (including circulating objects such as blood) simultaneously or sequentially.

Example 13: circulating biological subjects entering the blood or lymphatic vessels are treated by PBM.

1) Temporal variations in light power or irradiance may be performed to illuminate a circulating biological object passing near the light distributor over a range of fluence rates. The expression of irradiance and power given in example 4 can be adjusted to account for the different velocities of biological objects of blood or lymphatic vessels for which the PBM is directed.

2) The power of the light is synchronized with hemodynamic variables, such as flow changes due to heartbeat and vasomotor, to optimally illuminate the target biological object within the blood stream.

For example, in order to bypass the negative consequences of SARS-CoV-2, and more generally in the case of ARDS, to optimize the immune response, the hemoglobin oxygen affinity, the thrombotic process and promote tissue regeneration, especially by using the bystander effect of PBM by inserting one or more light distributors in the blood vessel (e.g. pulmonary artery), has shown impressive positive effects, as demonstrated by the inventors.

In particular, an example of a clinical procedure ("Seldinger method") for locating light distributors in the right and left pulmonary arteries is described below:

1. venous access was performed by right jugular vein puncture under ultrasound imaging using an i.v catheter (Surflo, terumo).

2. A 7Fr sheath (radiation focus, terumo) was introduced using the Seldinger method.

3. Under fluoroscopic guidance, a 4fr JR4 guide catheter (Cordis) was used, using a non-hydrophilic 0.035 inch guidewire (Terumo) to engage the right pulmonary artery ostium.

4. The 0.035 inch guidewire was removed and a hemostatic valve Y-connector was connected to the 4Fr JR4 guide catheter.

5. An optical distributor was placed between the upper right lobe and the lower right lobe pulmonary artery inlet through a 4fr JR4 guide catheter.

6. The 4Fr JR4 guide catheter was completely removed from the 7Fr sheath.

7. The 4fr JR4 guide catheter was flushed with saline through the hemostatic valve Y-connector.

8. Using the same procedure as described in figures 3, 4, 5 and 6, a second 4fr JR4 guide catheter was used to place a second light distributor between the inlets of the upper left and lower left pneumoconiosis arteries.

9. The 2 light dispensers (cannulated in the 4fr JR4 guide catheter) were attached to the skin using an adhesive system (Grip Lock, vygon).

As shown in fig. 20, the protocol may be adapted to perform PBM irradiation, wherein one optical splitter is placed in the atrium and the inferior and superior vena cava. The light distributor may be left for days or weeks to repeat the treatment periodically.

Example 14): combinations of different illumination schemes.

The PBM effect is caused by the absorption of light by different primary photoreceptors, particularly leading to a number of signal and transcription factor changes. PBM light is also known to photolyze NO from nitrosohemoglobin and to affect Nitrate Reductase Activity (NRA) which is involved in certain metalloproteins, which also releases unstable NO in the presence of hypoxia tension and nitrite. Since NO is recognized in mild or deep hypoxemia, different irradiation schemes must be considered to activate different mechanisms. For example, in circulating blood, a first lighting scheme may consist in delivering, at a suitable wavelength, a constant or pulsed irradiance/fluence rate (as high power as possible while avoiding thermal effects, i.e. typically several hundred mw.cm) applied during an optimal time (ranging from a few seconds to a few minutes) ^-2 ) The aim is for example photodissociation of nitrosylhemoglobin or of thiohemoglobin. This first irradiation scheme must be combined with a second scheme based on the hot spot concept described above.

Example 15: irradiation of biological objects can be performed by specific selection of hot spots, in particular for a duration of time Optimizing the treatment in this respect.FIG. 21 shows the use of the hot dotted line (10.+ -. 9,5 mW.cm) ^-2 The method comprises the steps of carrying out a first treatment on the surface of the 40 s). The figure depicts the fluence rate of illumination from a cylindrical dispenser to the myocardium at 689nm using a second class of bessel functions. Using this hot spot line, the first 40s period was 2.8mw.cm to a cylindrical dispenser ^-1 Is equal to 20mW.cm (corresponding to the surface of the distributor) ^-2 Is the first 3.5mm of fluence rate) that can be treated. Then 100mW.cm was used ^-1 The second period of 40s of power for treating tissue between 3.5 and 7 mm.

Example 16: irradiation of the biological object may be performed in synchronization with parameters corresponding to the plurality of hotspots.As shown in fig. 22, since the fluence rate decreases with increasing distance from the light distributor, when a high fluence rate (e.g. 15 or 25mW/cm ² ) In treating tissue proximate to the light distributor, the remote or deep tissue is exposed to a low fluence rate (e.g. 3mW/cm ² ). This can be used to optimize the treatment time as well as for specific parts of the biological object.

Example 17: using several PBM effective wavelengths exhibiting different penetration depths in tissue based on synchronization or sequence Treatment regimen. As shown in fig. 23, as fluence rate decreases with increasing distance from the light distributor, distant or deep tissue is treated with a penetration wavelength corresponding to the "hot spot" presented in fig. 4a and b for a fluence rate and duration, whereas tissue near the surface is treated with less penetration wavelength corresponding to the same "hot spot". This can be used to optimize the treatment time as well as for specific parts of the biological object.

Example 18: is a medical device for treating bone marrow and inducing cell lines designed for percutaneous introduction of bone marrow, strands Bone, tibia, iliac crest or other medullary area. Which includes one or more optical fibers. These fibers are placed in a catheter sheath that can be attached to the skin. The distal end of the optical fiber is sealingly connected to the catheter by an SMA connector connected to the source. The proximal end can be adjusted (between 2 and 8 cm) by retracting the catheter sheath, allowing for deployment of an optical fiber reinforced with a rigid material introduced into the spinal cord.

Example 19: increasing and homogenizing endogenous production of PpIX to improve photodynamic detection (PDD) and PDT performance. Embodiments of this surprising effect include:

1) Helmets with integrated light emitting diodes are used, which induce PBM irradiation through the skull over specific areas of the brain between 6 and 72 hours prior to PDD or PDT process, to treat brain cancers (including glioblastoma).

2)Increasing and homogenizing endogenous production of PpIX in plants and larvae. One example of such a method is in the agricultural field to increase the efficacy of phototoxic effects induced in grasses/larvae. It can be adapted to a variety of agricultural engines.

Example 20: triggering or spatial resynchronization of metabolic activity of biological objects. Spatial synchronization of metabolic activity is necessary to maintain local or systemic homeostasis and to allow blood flow in arterial or venous capillaries. Notably, the blood vesselsSynchronous local contraction from one location to another may cause vasomotor. These contractions can be seen as spatial wavefronts that move all the way along the blood vessel. For example, disruption of these synchronous contractions by impaired myogenic conduction may be responsible for many vascular lesions. Since it has been shown that different parts of a biological object can be targeted sequentially or consecutively by selecting specific hot spots, and since PBM can modulate or trigger specific metabolic activities, in particular within a myogenic frequency range, irradiation of damaged blood vessels, for example at specific distances (equal to the length defined by the spatial period of the contraction wave), can maintain the contraction from one location to another synchronous or trigger contraction from one location to another to restore blood flow in case of myogenic dysregulation.

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Claims (53)

1. An apparatus for applying photo-biological modulation (PBM) to a biological object, the apparatus comprising a light source delivering light at an appropriate temporal evolution of its optical power, the apparatus further comprising a processing and/or light control unit determining the appropriate temporal evolution of the optical power based on light delivery geometry on/in the biological object and biological object optical coefficients, characterized in that the processing and/or light control unit is further adapted to: continuously in each portion of the volume of the biological object, causing a PBM effect by generating one or more specific fluence rates during one or more specific times; the specific combined fluence rate and the timeThe parameters are selected from the following groups: at 180+Period 3 of 30s+2mW/cm ² Or at 80+25s period 11+9mW/cm ² Or at 40+20s period 16+10mW/cm ² Or at 15+Period of 10s 25+10mW/cm ² Or at 40+Period 10 of 1s+9,7mW/cm ² 。

2. The device according to claim 1, comprising a PBM monitoring and/or feedback system designed to adjust the optical power or irradiance based on the PBM effect.

3. The device of claim 1 or 2, comprising a glucose sensor, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence of the blood glucose level measured by the glucose sensor.

4. The device of any one of the preceding claims, comprising a cardiac output sensor, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence on the cardiac output measured by the cardiac output sensor.

5. A device according to any one of the preceding claims, comprising a krebs cycle enzyme kinetic measurement device, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence of the enzyme activity measured by the krebs cycle enzyme kinetic measurement device.

6. The device of any one of the preceding claims, comprising means for administering one or more exogenous agents to the biological subject.

7. The device according to any of the preceding claims, comprising a monitoring unit for PBM monitoring and/or feedback of PBM effects, the processing unit being adapted to adjust the optical power, the light delivery and/or the irradiance based on the PBM effects monitored by the monitoring unit.

8. The device according to any of the preceding claims, comprising a metabolic monitoring unit, the processing unit being adapted to adjust the optical power, the light delivery and/or the irradiance based on metabolic activity of the biological subject measured by the metabolic monitoring unit.

9. A device according to any one of the preceding claims, characterized in that the device is adapted to produce a combined use of wavelengths, at least one of which is inactive when used alone.

10. An apparatus that integrates the adjustment of radiometric and/or spectroscopic conditions used in PBMT based on frequency analysis of monitored parameters reflecting metabolic activity, particularly using wavelet theory.

11. The device according to claim 10, comprising a PBM monitoring and/or feedback system designed to adjust the optical power or irradiance based on the PBM effect.

12. The device of claim 10 or 11, comprising a glucose sensor, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence on the blood glucose level measured by the glucose sensor.

13. The device according to any of the preceding claims 10 to 12, comprising a cardiac output sensor, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence of the cardiac output measured by the cardiac output sensor.

14. The device according to any of the preceding claims 10 to 13, comprising a krebs cycle enzyme kinetic measurement device, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence of the enzyme activity measured by the krebs cycle enzyme kinetic measurement device.

15. The device according to any of the preceding claims 10 to 14, comprising means for administering one or more exogenous agents to the biological subject.

16. The device according to any of the preceding claims 10 to 15, comprising a monitoring unit for PBM monitoring and/or feedback of PBM effects, the processing unit being adapted to adjust the optical power, the light delivery and/or the irradiance based on the PBM effects monitored by the monitoring unit.

17. The device according to any of the preceding claims 10 to 16, comprising a metabolic monitoring unit, the processing unit being adapted to adjust the light power, light delivery and/or irradiance based on metabolic activity of the biological subject measured by the metabolic monitoring unit.

18. An apparatus predicts an optimal time to start application of PBM light based on a measurement of metabolic activity.

19. A device for performing PBM directly in blood contained in a cardiac compartment, pulmonary artery or vena cava.

20. The device of claim 19, comprising a PBM monitoring and/or feedback system designed to adjust the optical power or irradiance based on the PBM effect.

21. The device of claim 19 or 20, comprising a glucose sensor, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence on the blood glucose level measured by the glucose sensor.

22. The device of any of the preceding claims 19 to 21, comprising a cardiac output sensor, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence of the cardiac output measured by the cardiac output sensor.

23. The device according to any of the preceding claims 19 to 22, comprising a krebs cycle enzyme kinetic measurement device, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence of the enzyme activity measured by the krebs cycle enzyme kinetic measurement device.

24. The device of any one of the preceding claims 19 to 23, comprising means for administering one or more exogenous agents to the biological subject.

25. The device according to any of the preceding claims 19 to 24, comprising a monitoring unit for PBM monitoring and/or feedback of PBM effects, the processing unit being adapted to adjust the optical power, the light delivery and/or the irradiance based on the PBM effects monitored by the monitoring unit.

26. The device according to any of the preceding claims 19 to 25, comprising a metabolic monitoring unit, the processing unit being adapted to adjust the light power, light delivery and/or irradiance based on metabolic activity of the biological subject measured by the metabolic monitoring unit.

27. An apparatus for optimizing PBM dosimetry based on dynamic changes in distance separating an optical dispenser from a target biological fluid.

28. The device of claim 27, comprising a PBM monitoring and/or feedback system designed to adjust the optical power or irradiance based on the PBM effect.

29. The device of claim 27 or 28, comprising a glucose sensor, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence on the blood glucose level measured by the glucose sensor.

30. The device of any of the preceding claims 27 to 29, comprising a cardiac output sensor, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence of the cardiac output measured by the cardiac output sensor.

31. The device of any one of the preceding claims 27 to 30, comprising a krebs cycle enzyme kinetic measurement device, and wherein the processing and/or light control unit is adapted to adjust light dosage in dependence of the enzyme activity measured by the krebs cycle enzyme kinetic measurement device.

32. The device of any one of the preceding claims 27 to 31, comprising means for administering one or more exogenous agents to the biological subject.

33. The device according to any of the preceding claims 27 to 32, comprising a monitoring unit for PBM monitoring and/or feedback of PBM effects, the processing unit being adapted to adjust the optical power, the light delivery and/or the irradiance based on the PBM effects monitored by the monitoring unit.

34. The device according to any of the preceding claims 27 to 33, comprising a metabolic monitoring unit, the processing unit being adapted to adjust the light power, light delivery and/or irradiance based on metabolic activity of the biological subject measured by the metabolic monitoring unit.

35. A device that exhibits a variable longitudinal light emissivity profile to deliver optimal fluence rates in a moving biological object at different distances from the device light emitting surface.

36. The apparatus of claim 35, comprising a PBM monitoring and/or feedback system designed to adjust the optical power or irradiance based on the PBM effect.

37. The device of claim 35 or 36, comprising a glucose sensor, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence on the blood glucose level measured by the glucose sensor.

38. The device of any of the preceding claims 35 to 37, comprising a cardiac output sensor, and wherein the processing and/or light control unit is adapted to adjust the light dose in dependence of the cardiac output measured by the cardiac output sensor.

39. The device of any one of the preceding claims 35 to 38, comprising a krebs cycle enzyme kinetic measurement device, and wherein the processing and/or light control unit is adapted to adjust light dosage in accordance with the enzyme activity measured by the krebs cycle enzyme kinetic measurement device.

40. The device of any one of the preceding claims 35 to 39, comprising means for administering one or more exogenous agents to the biological subject.

41. The device according to any of the preceding claims 35 to 40, comprising a monitoring unit for PBM monitoring and/or feedback of PBM effects, the processing unit being adapted to adjust the optical power, the light delivery and/or the irradiance based on the PBM effects monitored by the monitoring unit.

42. The device according to any of the preceding claims 35 to 41, comprising a metabolic monitoring unit, the processing unit being adapted to adjust the light power, light delivery and/or irradiance based on metabolic activity of the biological subject measured by the metabolic monitoring unit.

43. A method for applying photo bio-modulation (PBM) to a biological object, wherein light is delivered in a suitable temporal evolution of optical power,determining the power based on the light delivery geometry on/in the biological object and the biological object optical coefficient, characterized in that the PBM effect is further induced by generating one or more specific fluence rates during one or more specific times in each part of the volume of the biological object in succession, the specific combined fluence rate and the time being selected from the following parameter group: at 180 +Period 3 of 30s+2mW/cm ² Or at 80+25s period 11+9mW/cm ² Or at 40+20s period 16+10mW/cm ² Or at 15+Period of 10s 25+10mW/cm ² Or at 40+Period 10 of 1s+9,7mW/cm ² 。

44. The method of claim 43, comprising the additional step of including the act of at least one exogenous stimulus.

45. The method of claim 44, wherein the exogenous stimulus is an agent.

46. The method of claim 44, wherein the exogenous stimulus is a temperature change.

47. The method of any one of claims 43 to 46, comprising the act of at least two exogenous stimuli, one of the at least two exogenous stimuli being an exogenous agent.

48. The method of any one of claims 43 to 47, wherein the optical power, optical delivery and/or irradiance are used to adjust the amplitude, phase and/or frequency of fluctuations of one or more parameters reflecting metabolic activity of the biological subject.

49. Use of the foregoing device or method as defined in any of the preceding claims for the treatment of Myocardial Infarction (MI) including acute myocardial infarction.

50. Use of a device or method as defined in any one of the preceding claims 1 to 48 for treating a biological subject suffering from ischemia and/or hypoxia/anoxia.

51. Use of a device or method as defined in any one of claims 1 to 48 for the treatment of Acute Respiratory Distress Syndrome (ARDS).

52. Use of a device or method as defined in any one of the preceding claims 1 to 48 for the treatment of metabolic activity disorders.

53. Use of a device or method as defined in any one of the preceding claims 1 to 48 for the treatment of disorders of insulin secretion.