WO2011050056A2 - Laser treatment of eye diseases - Google Patents

Abstract

A non-destructive pulsed laser beam is used to treat eye diseases in mammals, including diseases of retina and optical nerves, and to release heat shock proteins.

Description

LASER TREATMENT OF EYE DISEASES

RELATED APPLICATIONS

This application claims priority from a U.S. Provisional Patent Application No. 61/253,107, filed on October 20, 2009, which is incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

Diseases of the eye, especially degenerative diseases of the retina and optical nerve, represent a medical problem and a burden that will continue to increase with the aging of the global population. An estimated 230 million people in the world live with some kind of progressive degenerative eye disease. In the US, this number is close to 20 million and is estimated to triple by 2050. Three of the most prevalent degenerative eye diseases include glaucoma, which affects over 3 million in the US and almost 80 million globally; age-related macular degeneration (AMD), which afflicts 10 million in the US and almost 70 million in the world; and diabetic retinopathy, which affects 4 million in the US and 80 million globally.

While the burden of disease is significant, resulting in progressive blindness in many cases, therapeutic options are relatively limited. Several drug treatments exist for AMD, but only for the "wet" form that composes only 10% of all AMD cases. There is currently no approved drug treatment for dry AMD. While there are five different major drug treatments for glaucoma, for the most part the therapeutic effect is limited to controlling intraocular pressure. For normotensive glaucoma, which is much more prevalent than other forms, there is no treatment. Likewise, for retinitis pigmentosa, no satisfactory approved drug treatment exists.

Due to the unsatisfactory status of drug therapy for these diseases, a number of laser- based approaches have been tested to treat degenerative eye diseases. The majority of these involve the selective destruction or damage of eye tissue. For example, laser trabeculoplasty involves making small burns in the trabecular meshwork with a laser in order to open up drainage from the eye. Selective laser photocoagulation is used to stop bleeding of retinal blood vessels in AMD. Other treatments, such as grid photocoagulation for AMD, where patterns of small burns are made to the macula, have not proven to be effective. In general, acute laser therapy is designed to provide limited relief of specific symptoms of these diseases by selectively causing damage to or destruction of tissue. A variety of side effects may accompany destructive laser treatment of the retina, including loss of visual acuity, decreased visual field, loss of color vision, loss of night vision, decreased contrast sensitivity, pain, scarring, hemorrhage, and retinal detachment. A variety of adjustments to standard photocoagulation have been made to minimize the side effects of treatment.

More recently, a number of experiments have been performed with a subacute laser therapy approach called transpupillary thermotherapy (TTT). TTT has emerged as an approach to treatment of choroidal neovascularization (CNV) that uses prolonged delivery of milder thermal doses as opposed to short, high thermal doses that characterize laser photocoagulation. TTT involves the use of 810 nm continuous wave lasers that illuminate targets of about 1 mm in diameter with irradiances on the order of 10 W/cm² for 30-60 seconds. TTT has shown effectiveness in a number of small studies, but is still considered an investigational treatment. The largest clinical trial of TTT, the TTT4CNV Study, was a prospective, sham-controlled, randomized nationwide study that involved 22 centers and was conducted on 305 eyes of 305 patients with occult AMD. Two-thirds of eyes were treated and one-third received sham treatment. The preliminary results of the study did not show significant benefit of TTT for CNV over sham treatment. Of the patients who received TTT for CNV, 47% had modest or severe vision loss after two years, compared with 43% in those who received sham treatment. In addition to application to CNV, several investigators have examined the effect of TTT for neuroprotection in the eye. These studies, performed in animal models using acute damage to optical nerves (typically crush injury studies) have shown that TTT can significantly enhance the survival of neurons in the presence of injury (Kim et al. 2009; Ma et al. 2010). These studies have linked TTT thermal doses to the overexpression of heat shock protein 70 in the laser treated tissues, which can promote cytoprotection against neural injury (e.g., Park et al. 2001).

While TTT largely avoids the gross thermal damage that result from the use of destructive lasers, the treatment does raises retinal tissue temperatures to a significant degree (generally about 10° C) and results in histologically visible damage to retinal tissues. For experimental treatments with optical neuroprotection, high thermal temperatures were also generated in the treated tissues. This relatively high thermal dose, with its risk of collateral thermal damage, is not likely to represent a clinically useful treatment modality in diseases that will require chronic treatment.

For many degenerative eye diseases, effective treatments that can block the progression of cellular apoptosis and necrosis still do not exist. While destructive laser treatment plays a limited role in ameliorating symptoms of these diseases, nondestructive laser treatments have proven to be only mildly effective and limited by associated unwanted thermal damage. A useful laser treatment needs to be able to mobilize cytoprotective elements in treated tissues such as HSP70 without the accompanying, unwanted thermal damage.

SUMMARY OF THE INVENTION

The invention provides methods of administering a non-destructive laser treatment to a subject with a disease condition to improve the disease condition treated. The invention further provides methods of treating diseases of the eye with a nondestructive laser.

The invention further provides methods for treating diseases of the eye by causing both the release of significant amounts of heat shock proteins from inside cells within the retinal tissues and the significant overexpression of heat shock proteins through exposing these tissues to a non-destructive laser treatment. In the methods of the invention, the temperature of the laser-treated tissue is raised to at least 39°C but maintained at or below 42°C. The method of the invention does not involve tissue damage caused by excess heating of the tissue that results in either apoptosis or necrosis of more than 1% of the cells in the treated tissue, or the disruption of the normal architecture or function of the laser-treated tissue.

In the methods of the invention, the laser operates in a pulsed manner. The pulse duration is about 100 picoseconds (ps) to about 100 nanoseconds (ns), about 500 ps to 50 ns, or about 1 to 20 ns. The frequency of the laser is about 1 Kilohertz (Hz) to about 100 kilohertz (kHz), about 1 to about 50 kHz, about 10 to about 30 kHz.

In the methods of the invention, the average power of the laser is typically below 1 Watt, the pulse energy of the laser is below 1 mJ, and irradiance of the laser is less than 50 Watt/centimeter² (W/cm²) at all wavelengths.

In the methods of the invention, treatment durations of the laser are about 10 seconds (s) to about 2 minutes (min), or about 30 s to 60 s. Treatment may involve a single laser dose or an indefinite series of doses repeated about every two to fourteen days.

The invention provides methods that can be practiced using near infrared lasers that can increase the release of and overexpression of heat shock proteins from within laser treated cells without causing damage to or destruction of the laser-exposed cells or tissues, or to cells or tissues close to the site of laser exposure. Lasers for use in the methods of the invention emit light in the near infrared range, with wavelengths of about 750 nanometers to 1200 nanometers. Appropriate lasers for the method of the invention can include but are not limited to titanium-sapphire (670-1130 nm), Alexandrite (700-800 nm), gallium-aluminum- arsenide (750-850 nm), chromium-fluoride (780-850), indium-gallium arsenide (904-1065 nm), neodymium-doped yttrium lithium fluoride (1047 and 1053 nm), neodymium-doped yttrium aluminum garnet (1064 nm), and helium-neon (1153 nm). Laser wavelengths for use in the methods of the invention include, but are not limited to 750 nm, 810nm, 850 nm, 930 nm, 950 nm, 980 nm, 1047 nm, 1064 nm, and 1 153 nm.

In the methods of the invention, the treatment areas on the retina are about 1-20 mm across, 1-10 mm, or 3-5 mm in diameter. The pulsed laser energy can be applied to this treatment area in a simultaneous manner or in an incremental manner using a line of 1-20 mm in length that is scanned back and forth across the treatment area or a focused spot that is scanned across the treatment area.

The invention provides methods for altering the exposure of the light so that the laser intensity profile is relatively uniform with a variation in energy of 50% or less from edge to center, or completely uniform at every point in the treatment area.

The methods of the invention include treating chronic degenerative diseases of the retina wherein the diseases are associated with the disruption and death of retinal cells such as astrocytes, cone cells, glial cells, Miiller cells, neurons, rod cells, or retinal pigemented epithelial cells. Diseases of particular relevance include degenerative diseases such as glaucoma, age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, autosomal dominant drusen, choroidal neovascularization, cystoid macular edema, ischemic retinopathies, Malattia Leventinese, retinal dystrophy, retinoschisis, Stargardt's disease, and vitelliform macular dystrophy. The method of the invention also includes treatment for acute inflammatory diseases of the retina and optic nerve such as papillary optical neuritis, neuromyelitis optica, inflammatory optic neuropathy, and acute retinitis.

The method of the invention also includes treatment and prevention of acute ischemic or hypoxic conditions of the retina or optical nerve such as ischemic injury following stroke or ischemic-perfusion injury that result of surgery.

In the methods of the invention, an increase in a detectable response to treatment leads to the improvement of the disease condition without the occurrence of significant damage to or destruction of the treated tissue, including the development of fibrosis or scarring. Improvement can include disease remission, amelioration of symptoms, or decrease in morbidity or mortality.

In the methods of the invention, the use of the specific parameters of wavelength, laser pulse duration, pulse frequency and duration of laser exposure allow for an

improvement in the disease condition at a lower treatment irradiance and temperature level in the treated tissue compared to a laser of identical wavelength not operating at the specified pulse duration and pulse frequency.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram showing the effect of thermal containment on temperature gradients within RPE tissue and neighboring neuronal tissue subjected to different pulse durations of 514 nm laser light. Spatial temperature profile of the RPE and the neural retina in the laser beam axis after irradiation with a 5.5-μΙ short (5-μ8) laser pulse (threshold of single pulse). Dashed line shows the temperature profile after a long (1-s) pulse at threshold power (11 mW). Arrowhead indicates the location of the "reference point" as described in the text. A sharp temperature gradient is observed at the end of a short pulse. The short-pulse temperature profile is repetitively built up every 2 ms. (Roider et al. 1993) Figure 2 is a graph showing the inverse relationship between temperature and exposure time for the development of tissue damage. Rates of accumulation of damage in tissue based on the rate coefficients. Times associated with dotted lines indicate the number of seconds to achieve threshold damage for a step rise ion tissue temperatures. (Welch 1984) Figure 3 is a series of plots showing the relationship between temperature and the onset of apoptosis and necrosis over time in tissue cultures of murine mastocytoma cells. (Harmon et al. 1990)

Figure 4 shows the difference between the shape of the acoustic waves generated by long pulse lasers (no stress containment) and short pulse lasers (stress containment)

(Esenaliev et al. 1993).

Figure 5 shows an illustration of the portions of apoptotic signaling pathway downstream of the mitochondria that are inhibited by heat shock protein 70. (Giffard et al. 2008)

Figure 6 shows a diagram of the activation pathway of protein kinase C, which is initiated by G protein coupled receptor signaling and mediated by phospho lipase C. G-protein coupled receptors (GPCRs) transduce a variety of signals from the extracellular environment across the plasma membrane. One of the common signaling systems utilized by GPCRs activates protein kinase C (PKC), a ubiquitous family of serine/threonine protein kinases. The pathway leading to PKC activation starts with a class of GPCRs that interact with and activate Gq G-proteins when the receptor has agonist ligand bound. GPCRs that act through Gq include some muscarinic acetylcholine receptors, many peptide receptors, and the 5-HT2 serotonin receptors. Activated Gq with GTP bound activates its downstream target phospholipase C (PLC) to hydrolyze the membrane lipid PIP2, producing IP3 and

diacylglycerol (DAG). IP3 is water-soluble and diffuses through the cytoplasm to the ER, where it binds to and opens a calcium channel, releasing calcium stores from inside the ER into the cytoplasm. Calcium alters many cellular processes, in part by binding to regulatory proteins such as calmodulin and calcineurin. The interaction of both DAG and calcium with PKC activates its kinase activity and the phosphorylation of many different protein targets alters their activity. The involvement of PKC in cellular proliferation and the cell cycle is indicated by the activity of tumor promoters like phorbol esters as PKC activators.

(http://www.biocarta.com/pathfiles/h_PKCPATHWAY.asp)

Figure 7 shows a graph comparing ocular transmission and retinal tissue absorption of laser light in the visible and near IR portion of the electromagnetic spectrum.

DETAILED DESCRIPTION OF THE INVENTION

As used herein "treating" a disease in a subject or "treating" a subject having a disease refers to subjecting the subject to a laser exposure, in such a manner that the extent of the disease is decreased or prevented. For example, treating results in the reduction of at least on sign or symptom of the disease or condition. Treatment includes (but is not limited to) administration of an exposure to a laser as described herein subsequent to the initiation of a pathologic event. Treatment can also include administration of an exposure to a laser in order to prevent the onset of the sign or symptoms of the disease or condition. Treatment can require administration of an agent and/ or treatment more than once.

As used herein, a "condition" includes any abnormality that can occur in a subject including any disease, infection, disorder, tumor, cancer, inflammation, or change in cellular structure and function.

As used herein, "destructive" is understood as causing either significant apoptosis or necrosis of the cells in a tissue, or any disruption of the tissue itself. As used herein, "nondestructive" is understood as not causing either significant apoptosis or necrosis of the cells in a tissue, or any disruption of the tissue itself. As used herein, "apoptosis" refers to the regulated death of a cell or, collectively, a tissue. Apoptosis is often referred to as programmed cell death because it is a genetically- regulated process that involves a series of intracellular signaling cascades. It is a normal part of the cell life cycle. Apoptotic cells are characterized by membrane blebbing, cell shrinkage, condensation of chromatin, and fragmentation of DNA followed by rapid engulfment of the corpse by neighboring cells or other phagocytic cells. Unlike necrosis, apoptosis does not result in the significant release of the contents of the cell into the extracellular space and therefore does not trigger inflammatory responses.

As used herein, "necrosis" refers to the chaotic, uncontrolled, and non-programmed death of a cell or, collectively, of a tissue. Necrosis occurs when irreversible exogenous injury occurs to a cell leading to the severe disruption of the cellular membrane and release of the cellular contents. Necrosis is characterized by impairment of the cell's ability to maintain homeostasis leading to an influx of water and extracellular ions into the cell, vacuolization of the cytoplasm, breakdown of the cellular DNA, and organelle membrane and cellular membrane rupture. The ultimate breakdown of the cell membrane releases the contents of the cell, including lysosomal enzymes, into the extracellular space. As a result, necrosis often results in inflammatory response. Significant necrosis refers to the number of cells that are killed within a tissue, expressed either as a ratio (percent) of the cells exposed to the laser, as a defined number of cells per laser exposure, or a defined area of cells. Significant necrosis is uncontrolled cell death of at least 1% of the cells.

As used herein, "tissue disruption" is understood as the physical separation, fragmentation, removal, or disaggregation of cells within a tissue, or of one layer of tissue from another.

As used herein, "retina" refers to the light-sensitive membrane lining the back two- thirds of the inner eyeball and connected by the optic nerve to the brain. The retina is a relatively thin structure, about half a millimeter in depth. It integrates a complex of photosensitive cells (rods and cones) and neurons converting light into electrical signals to the brain. The photoreceptors and nerve cells are supported by a number of other cells such as the retinal pigmented epithelial cells and the Miiller cells that provide structure to the retina. The retina also includes a network of blood vessels that feed this complex. There are ten layers in the retina. These are, from innermost to outermost: 1) the inner limiting membrane, consisting of the Miiller cell footplates; 2) the nerve fiber layer, consisting of the ganglion cell nuclei; 3) the ganglion cell layer, consisting of the nuclei of ganglion cells; the inner plexiform layer, consisting of fibrils arising from the ganglion cells and axons of the inner nuclear cells; 4) the inner nuclear layer, containing bipolar cells, horizontal cells, and amacrine cells; 5) the outer plexiform layer (known in the macular region as the fiber layer of Henle), consisting of rod and cone axons, horizontal cell dendrites, and bipolar dendrites; 6) the outer nuclear layer, consisting of the cell bodies of the rods and cones; 7) the external limiting membrane, which separates the inner segment portions of the photoreceptors from their cell nuclei; 9) the photoreceptor layer, containing the outer segments and inner segments of the rod and cone photoreceptors; and 10) the retinal pigment epithelium. The key anatomic elements of the retina include the macula (an oval shaped, pigmented area near the center of the retina that represents a major concentration of photoreceptors), the optical disc (where the optical nerves bundle and leave the retina), and the network of blood vessels that feed the retinal cells.

As used herein, "optic nerve" refers to the nerve that carries signals from the eye to the brain. The optic nerve originates within the retina, where millions of nerve fibers converge and then exit the eye toward the brain. The area where these nerve fibers converge is known as the optic nerve head. Degenerative eye diseases that affect the optic nerve mainly cause loss of neurons in the optic nerve head. As used herein, "subject" refers to a mammal. A human subject can be known as a patient.

As used herein, "mammal" refers to any mammal including but not limited to human, mouse, rat, sheep, monkey, goat, rabbit, hamster, horse, cow or pig. A "non-human mammal," as used herein, refers to any mammal that is not a human.

As used herein "exposure" may mean treatment with a laser for a time useful to the invention. In one embodiment, exposure means to treat with a laser applied in a pulse, wherein the pulse is applied for a particular duration. The range of pulse durations are in the fraction of nanosecond to hundreds of nanoseconds (for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nanoseconds). It is understood that the actual pulse length will vary somewhat based on the limitations of the laser and the switching rate/ shutter speed. In another embodiment, "exposure" means to treat with a laser of a particular pulse repetition (pulse frequency).

Optimal pulse frequencies range from about 1 kilohertz (kHz) to about 100 kHz (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kHz). It is understood that the actual pulse frequency will vary somewhat based on the limitations of the laser and the switching rate/ shutter speed.

"Exposure" may also mean treatment with a laser of a particular wavelength where the range of wavelengths is in the near infrared portion of the electromagnetic spectrum (approximately 750 nm to 1200 nm, for example, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 960, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, or 1200 nm). Relevant wavelengths in common use in medical practice are 810, 830, 930, 950, 1040, 1064, 1100, and 1153 nm. In another embodiment, "exposing" means to expose a subject to a laser with a particular peak energy, where the range of pulse energy is less than 1 milliJoule (1 x 10^~3 J) and equal to or more than 1 microJoule (1 x 10^~6 J) (for example, 1, 10, 20, 30 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 microJoules).

"Exposure" may also mean treatment with a laser of a particular power density or irradiance, where the range of irradiance is measured at the tissue 1 to 50 W/cm² (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 W/cm²).

"Exposure" may also mean treatment with a laser for a particular length of time. The range of exposure times can be about 10 seconds to about 120 seconds (for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 seconds).

"Exposure" may also mean treatment with a laser of a particular area of the retina or optical nerve. Typical treatment areas are about 1-20 mm in diameter (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 mm). The treatment area is typically round, since this is one of the fundamental geometries of laser beams, but could also be rectangular if a scanning linear beam is used. Treatment may involve exposure of multiple areas of the subject during a single treatment, or progressive treatment of individual areas within the retina. In a particular embodiment, the entire retinal field of the eye can be treated in a simultaneous or progressive fashion during a single treatment.

As used herein, a "laser" refers to an electronic-optical device that emits coherent light radiation. A typical laser emits light in a narrow, low-divergence monochromatic (single-colored, if the laser is operating in the visible spectrum), beam with a well-defined wavelength. In this respect, laser light is in sharp contrast with such light sources as the incandescent light bulb, which emits light over a wide area and over a wide spectrum of wavelengths.

As used herein, a "laser" includes any laser that is currently available or may become available that can provide the appropriate pulse duration, power, and pulse frequency required by the methods of the instant invention. Currently available lasers that can be used in the methods of the invention include, but are not limited to gas vapor lasers, metal vapor lasers, pulse dye lasers, solid state lasers, semiconductor lasers and fiber lasers. Examples of lasers that can provide appropriate pulse duration, power density, and pulse frequency include a Q-switched Alexandrite laser at 755 nm, a Q-switched 810 nm diode laser, a pulsed fiber laser such as the IPG Photonics YLP series ytterbium pulsed fiber laser at 1055-1075 nm, or a Q-switched neodymium-doped yttrium aluminium garnet (Nd:YAG) laser such as the RMI 15 Q-Switched Diode-Pumped Solid State Laser operating at 1064 nm.

As used herein, a "heat shock protein" refers to a type of protein that is important to cell protection and whose expression is typically increased by cells when they are exposed to elevated temperatures or other stresses. Heat shock proteins (HSPs) are named according to their molecular weights. For example, HSP27, HSP60, HSP70 and HSP90 (the most widely- studied HSPs) refer to families of heat shock proteins on the order of 27, 60, 70 and 90 kiloDaltons in size, respectively. There are five major groups of HSPs found in humans: 20- 30, 50-60, 70, 90, and 100-110 kDa. Some HSPs, such as HSP70, are found in virtually every organism and its structures are highly conserved across species. The small 8 kDa protein ubiquitin, which marks proteins for degradation, also has features of a heat shock protein. Other small HSPs such as B crystallin are important in preservation of eye tissue cells such as retinal pigment epithelial cells under stress and its quantities are also increased under stress.

As used herein, "expression" refers to the process by which genetic sequences are transcribed into the production of protein, including post-translational folding.

"Overexpression" refers to a gene -regulated production of excess amounts of a protein compared to the normal baseline quantities. The protein expression can be determined by techniques such as Western blotting, fluorescence microscopy of green fluorescent labeled proteins, or enzyme-linked immunosorbent assay (ELISA). As used herein, "hyperthermia" refers to a condition of elevated temperature or a process of elevating the temperature in a cell or tissue above normal by exposing the cell or tissue to a source of energy that results in the generation of heat within a cell or tissue or the transfer of heat into a cell or tissue. As used herein, "laser hyperthermia" is the use of a laser to produce heating of cells or tissue. Hyperthermia may be produced by direct exposure of a cell or tissue to the laser or by transfer of heat from an adjacent cell or tissue area. As used herein, "microhyperthermia" refers to transient elevation of temperatures in very small (typically intracellular) volumes.

As used herein, "release" refers to the externalization of substances that are normally resident within the cell or at the cell membrane. Release may occur through an active process or through a passive process using a variety of mechanisms, which may include but are not limited to ion pumps, protein chaperones, exosome or lysosome release, lipid raft release, membrane blebbing, gap-junction transfer, osmosis or diffusion. Significant release refers to externalization of at least 10% of any specific population of a specific substance within the cells of the laser exposed tissue

As used herein, a "decrease" as it refers to a diminution in the level of a response as defined herein, means a response that is at least about 2-fold (for example about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1000-fold or more) or at least about 2% (for example, about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100%), less than the level of response of an untreated subject, for example a subject that has not been exposed to a laser.

As used herein, a "detectable response" includes a discernable, preferably a measurable level of a response that occurs in a subject that has been exposed to a laser, as described herein, but not in a subject that has not been exposed to a laser. A "response" that is detected includes, but is not limited to, one or more of an increase or decrease in HSP levels, a decrease in the level of hyperthermia required to produce a therapeutic response, or a decrease in the rate of cellular apoptosis due to a degenerative disease condition.

As used herein, "measuring" means detecting or determining the amount, for example, an increase in the release or expression of HSPs or an increase or decrease in temperature. Measuring is the steps taken to determine if an increase or decrease in a level of the material to be detected. Measuring may indicate a level that is zero or below the level of detection or greater than the linear detection limit of the method used for measuring.

Measuring according to the invention is performed in vitro or in vivo, for example in the retina at the site of laser exposure, in serum or in blood or other biological sample, tissue or organ.

"Measuring" also means detecting a change that is either an increase or decrease in the response (for example an increase or a decrease in cellular necrosis or apoptosis, a decrease in the level of hyperthermia required to produce a therapeutic response, an increase in the release of HSPs, an increase in the level of HSPs in the tissue, a decrease in the size or mass of diseased tissue, or the death rate from a disease) of a subject to treatment with a laser, according to the methods described herein.

Measuring is performed in a subject wherein said subject has been exposed to a laser. Measuring is also performed in a control subject, for example a subject that has not been exposed to a laser.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

As used herein, "about" is understood to be relative to the amount of variance typically tolerated in the specific assay, method, or measurement provided. For example, "about" is typically understood to be within about 3 standard deviations of the mean, or two standard deviations of the mean. About can be understood as a variation of 20%, 15%, 12%, 10%), 8%), 5%o, 3%o, 2%o, or 1%, depending upon the tolerances in the particular art, device, assay, or method.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

The invention provides a method of treating and preventing diseases of the retinal and optical nerve tissues with a non-destructive laser. The laser enhances the protection of retinal and optical nerve cells by increasing the level of heat shock proteins in treated cells, particularly HSP70. In addition, the method of the invention is significantly more efficient in generating higher levels of HSP70 in treated cells than other non-destructive laser treatments that are currently used, such as TTT, or those described by practitioners of the art, thus enabling a significant reduction in the temperature of the treated tissues and the total thermal dose required to adequately increase heat shock protein 70 to protective levels. This makes the laser much safer for treatment of the eye, especially for chronic treatment, than existing or described ophthalmologic laser systems. Without wishing to be bound by mechanism, it is suggested that laser treatment produces more efficient increase of HSP70 in treated tissues in two major ways. First, it alters the scale at which thermal and acoustic energy is diffused in treated tissue to the intracellular level, which accentuates the thermal and acoustic stress effects of the laser on treated cells while limiting the possibility of thermal damage. Second, the parameters of laser treatment are sufficient to stimulate the release of HSP70 contained within reservoir cells of the retina and optic nerve. This extracellular release of baseline quantities of HSP70 makes this cytoprotective protein available to surrounding cells that lack HSP70, and increases the level of HSP70 subsequently generated by these reservoir cells.

The increased efficiency with which laser energy affects cells where it is absorbed, coupled with the dynamic of HSP70 release, leads to a significantly higher level of HSP70 expression in the treated tissue than would result from an equivalent thermal dose of another laser that does not possess these specific parameters. These gains in efficiency of converting light energy into protein expression mean that a medically effective treatment can be given using smaller laser irradiances with significantly lower overall temperature in the treated tissue, as well as reduced thermal doses. This reduction in irradiance and lower resultant tissue temperature means that treatment can be given while avoiding any significant apoptosis, necrosis or disruption of the treated tissue that characterizes current acute and subacute (also referred to as "subthreshold") laser treatment approaches. As a practical result, medically effective laser treatment of the retina and optical nerve can be made far safer, especially for repeated chronic treatment of degenerative retinal diseases.

These effects require a laser emitting light at a specific combination of wavelength, pulse duration, pulse frequency, pulse energy, irradiance and treatment area. Lasers with the appropriate combinations of wavelength, pulse duration, pulse frequency, pulse energy, irradiance and treatment area will result in therapeutically more effective treatment at a given dose than lasers that do not have the appropriate combination of parameters. Principles of Laser Thermal and Stress Confinement

The biological effects of lasers are based on the conversion of laser light energy into other types of energy within a cell or tissue. The principle conversion form of energy is heat, but lasers can also lead to generation of acoustic energy, as well as chemical, other forms of electromagnetic energy, and specific types of kinetic energy. Only the first two types are discussed here.

Energy conversion of laser light is mediated by chromophores, which are chemical compounds capable of selective light absorbance. Examples of chromophores are DNA, melanin, hemoglobin, cytochrome c, collagen, and water molecules. In retinal tissues, important chromophores (apart from water) are shown in Table 1. For the most part, these chromophores are most sensitive to the light in the ultraviolet and visible light range. In the infrared range, melanin and hemoglobin have limited absorption, while water becomes increasingly important.

Table 1: Representative Retinal Chromophores and Key Absorption Spectra

Chromophore Main Location Absorption Spectra

Melanin RPE* and choroid 400-1000 nm

Xanthophyll Inner/outer plexiform layers 420-500 nm

Photo pigments Photoreceptor outer segments 420-780 nm

Lipofuscin RPE (Not specified)

Hemoglobin Choroidal and retinal vessels 450 and 550 nm

*RPE = retinal pigment epithelium

(Newsom et al. 2003)

Conversion of laser photons into other forms of energy occurs at a molecular level. In most cases, the majority of light energy is converted into thermal energy. This is especially the case for infrared lasers, where absorption by a limited number of chromophores such as melanin, hemoglobin and water, dominate. Under specific conditions, thermal energy subsequently generates acoustic effects in the tissue. Nevertheless, the scale of the effect of these thermal and acoustic effects depends on the duration of the laser pulse absorbed. The scale at which thermal and acoustic effects effect laser treated tissues differ based on the speed at which heat and sound are respectively transmitted through the tissue.

Once laser energy is converted into heat via a chromophore, this heat begins to dissipate into the surrounding tissue. Traditionally, analysis of heat transfer by conduction in biological tissues is performed using Fourier's law, where heat conduction is assumed to be an instantaneous process with an infinite speed of propagation of the thermal signal, indicating that a local thermal change causes an instantaneous perturbation in the temperature at each point in the medium. This model does not apply in laser irradiation, where generation of heat occurs in individual chromophores and is then propogated through the tissue. A damped- wave heat conduction model is more relevant in this case, based on the concept of thermal relaxation time (t_r), defined as the time it takes for 50% of the heat to diffuse out of a specific volume. The larger the volume, the longer it takes for the thermal energy to dissipate and the longer the thermal relaxation time. The rate of heat dissipation is a property of the thermal diffusion constant of a tissue a (the symbols κ, D, and k are also used), determined by several variables: the thermal conductivity, the density, and heat capacity of a laser- irradiated substance. For water at 37° C, a = 0.15 mm²/second. Based on a, the thermal relaxation time of a specific volume can be described by the equation t_r = 1/2αμ², where 1/μ is the diameter of the volume of diffusion. Table 2 shows approximate thermal relaxation times for important biological structures. Table 2: Approximate Thermal Relaxation Times for Important Biological Structures

Structure Size t_r

Large blood vessel 200 μΜ 20 msec

Small blood vessel 50 μΜ 5 msec

Melanosome 0.5-1 μΜ 1 μβεΰ

Protein 5 nm lOO sec

While the chromophores can affect the specific tissue targets of laser absorption, pulse duration defines the volume over which thermal effects are concentrated. When heat is generated by the chromophore at a much faster rate than it can be dissipated to the surrounding tissue, the result is a significant increase in the temperature within a local region. This condition is called thermal containment, and is considered to be met when t_p ~ 0.25t_r. Thermal containment results in the development of a highly localized temperature gradient. The magnitude of the temperature rise in the thermally confined volume is then a function of the pulse energy of the laser.

The phenomenon of thermal containment means that, as pulse durations decrease, the scale of thermal effects are reduced to increasingly smaller dimensions. At similar pulse energies, temperature rises are confined to smaller volumes, while overall heat dissipation grows more limited. Figure 1 shows an example of this with retinal pigment epithelial (RPE) tissue that is subjected to varying pulse durations of a 514 nm laser. As the pulse duration decreases to the microsecond range, increasingly large temperature gradients are created in the regions around the melanin granules in the RPE cells, while less heat is dissipated into the surrounding neuronal tissue.

While pulse duration accounts for the localization of the temperature gradient within the zone of thermal containment and pulse energy determines the magnitude of the temperature increase, the biological effects of photothermal energy in laser treated tissues also depend on total thermal dose (temperature x time) and not solely of the temperature alone. There is an inverse relationship between the temperature and the duration of exposure needed to produce similar biological effects in tissue. A 50° C increase in temperature for a microsecond may not irreversibly harm a cell, but a one minute exposure to a 20° C rise will kill it. This relationship between temperature and exposure time with respect to tissue damage is illustrated by Figure 2. In general, for sustained doses, raising tissue temperatures to more than 43° C results in dramatically increased apoptosis and necrosis. Above 43° C the rate of cell killing doubles for every degree centrigrade (Jeong et al. 2003). Figure 3 illustrates the temperature-dependent nature of both apoptosis and necrosis in heat-treated cells.

At sufficiently high temperatures (typically above 56° C), proteins in cells and tissues are significantly and irreversibly damaged with exposures longer than one second, leading to the loss of cellular function and resulting in the triggering of necrosis. Irreversible cell alteration occurs when the kinetic energy caused by heat overcomes the weak hydrogen bonds and van der Waals interactions that help maintain the three-dimensional structure of proteins, causing them to denature. Exposure of tissues to lasers of sufficient power can result in necrosis of not only of a diffuse populations of cells within the tissue, but of the entire laser-exposed tissue. At high enough power levels tissues can be coagulated or carbonized by a laser.

At wavelengths preferentially absorbed by water, short laser pulses of sufficient energy can vaporize the water in and around a cell before protein denaturation can take place, leading instead to cellular and tissue disruption. At very short laser pulse durations (typically in the picosecond range) even modest amounts of pulse energy can cause the heating of small volumes of water to very high temperatures, well above 100°C. When this occurs, the water experiences a rapid phase change into a gas, referred to as a phase explosion. This phase transition results in the generation of extremely hot vapor bubbles inside or outside the cell (microcavitation). The subsequent rapid expansion of these vapor bubbles disrupts cell organelles or cell membranes, leading to the cell death. On a larger scale, this effect can cause rapid vaporization or disruption of the tissues, in a process called photoablation. In these cases water is the primary chromophore, and photoablation can occur even without laser pulsing, simply by rapidly heating the water n the exposed tissue to the point of phase explosion.

Below the level of microcavitation and photoablation, short pulse lasers can generate non-damaging acoustic effects in cells or tissues as a result of differential thermal expansion in the laser treated tissue. Continuous wave laser irradiation of the tissue typically results in a constant flow of heat out of the treated tissue, resulting in a temporally consistent thermal gradient in the tissue. However, if the laser light is pulsed, thermal discontinuities occur between the focus of laser absorption and the surrounding tissue, resulting in different rates of thermal expansion and thus pressure differences between the chromophore absorbing laser light and the surrounding tissue. The pressure difference generates an acoustic wave that propogates at a much slower rate than that of heat dissipation. The speed of sound in tissue, σ, is about 1500 M/sec. The time needed for an acoustic wave to move out from a volume of tissue with a specific diameter δ is t_a = δ/σ. The condition of stress containment is achieved when t_p < t_a. Table 3 shows the approximate stress containment parameters for important biological structures.

Table 3: Approximate Stress Containment Times for Important Biological Structures Structure Size t_a

Large blood vessel 200 μΜ 0.1 msec

Small blood vessel 50 μΜ 30 μβεΰ

Cell 10 μΜ 5 nsec

Melanosome 0.5-1 μΜ 500 psec

Protein 5 nm 3 fsec

Another important factor in laser-induced acoustic effects is the rise time of the pulse. Long laser pulses tend to be characterized by gradual increases in the energy of the pulse, while short laser pulses have much sharper rise times. At very short pulse durations, pulse rise times are almost insignificant, resulting in very sharp thermal discontinuities that propogate acoustic waves with similarly sharp fronts (Figure 4).

For the most part, pulse durations above a millisecond do not generate significant acoustic effects in tissue and their effects are further diminished by harmonics and acoustic scattering in the tissue. However, as the duration of a laser pulse becomes very small (below a microsecond), the volume of the "stress confinement" significantly decreases and the "leading edge" of the acoustic stress wave grows sharper. In the nanosecond range, laser generation of acoustic waves can generate measurable stress waves in tissues that can induce biologic responses. The magnitude of the acoustic stress wave increases as the energy of the laser pulse increases. In human tissues with laser pulses in the visible to mid-infrared range, thermoacoustic effects becomes significant at pulse durations below 100 nanoseconds. As pulse durations shorten into the picosecond range, the thermal rise times are generated in such small volumes that thermoacoustic effects give way to phase explosion and

microcavitation with subsequent cell damage. Both laser thermal and laser thermoacoustic effects can, at sufficiently short pulse durations or sufficiently high energy levels, lead to cellular apoptosis, necrosis, or tissue disruption. Cellular apoptosis and necrosis may occur immediately upon exposure to the laser or over a longer period of time depending on the power of the laser and the energy of the pulse used. Tissue disruption from photothermal or photoablative effects is typically immediate.

Measurement of apoptosis can be detected by practitioners of the art using flow cytometry techniques. For example, apoptotic cells show an increased uptake of the vital dye H0342 compared to live cells due to a changes in membrane permeability. Apoptosis can be measured using a number of other assay-based approaches including measurement of DNA fragmentation, membrane phospholipid changes, interleukin-lbeta converting enzyme-like protease activation, or nucleosomal fragmentation by DNA agarose gel electrophoresis.

Finally, apoptosis can be detected through visual means such as changes in cell morphology Measurement of the concentration of necrotic cells in a particular tissue or culture is routine in the art. Methods and kits are known in the art to detect necrosis. The number of cells undergoing necrosis can be readily scored and expressed as a percentage of cells exposed to the laser. Practitioners in the art are able to easily distinguish between necrotic death and apoptotic death of cells. Necrotic cells can be detected by flow cytometry techniques, such as the addition of the nucleic acid stain PI, which binds to DNA or RNA but cannot permeate cell membranes and therefore is visible under fluorescence only if the cell membrane has been compromised, or stains such as propidium iodide or 7-AAD that discriminate cells which have lost membrane integrity. In addition, they can be visualized optically using standard staining and microscopy techniques.

Measurement of tissue disruption is routine in the art. Disruption can be assessed visually by means of naked eye inspection, use of magnifying glasses, biopsying of the tissue, preparation of slides and microscopic inspection of histology. Staining may be used to help differentiate tissue types in the slide.

The method of this invention utilizes laser irradiation of retinal and optic nerve tissue that generates a photothermal effects and thermoacoustic effects in the treated tissue that is biologically significant and leads to therapeutic benefit while avoiding significant cellular apoptosis or necrosis or any tissue disruption. Preferably less than 1% of cells are

irretrievably damaged upon laser exposure using the methods of the invention and the tissue is not disrupted in any way.

In one embodiment, cellular apoptosis, necrosis and tissue disruption is prevented by limiting the thermal dose in the cells exposed to the laser in the methods of the invention to below the levels that lead to cellular apoptosis or necrosis. In another embodiment, the overall temperature of the treated tissue is maintained at or below 42° C. In another embodiment, significant damage or disruption is prevented by limiting the pulse duration of the laser to above one nanosecond. In another embodiment, significant damage or disruption is prevented by limiting the pulse energy to below 1 mJ. In another embodiment, significant damage or disruption is prevented by limiting the irradiance to below 50 W/cm². In another embodiment significant damage or disruption is prevented by limiting the power of the laser to less than 1 Watt of average power. In yet another embodiment, significant damage or disruption is prevented by limiting the wavelength to the range of 750 nm to 1200 nm.

Wavelengths below 750 nm are visible and can have adverse effects in patients' eyes since the treatments are delivered over a period of many seconds or even a minute. Wavelengths above 1200 nm are also avoided because at longer wavelengths water absorption becomes significant and can result in unwanted heating of the lens, aqueous and vitreous humor.

The parameters of high-frequency repetition of nanosecond pulses of near infrared laser energy at lower powers and irradiance result in the generation of non-damaging, small- scale thermal effects ("microhyperthermia") and subsequent thermoacoustic effects that together have a therapeutic benefit in treated retinal and optical nerve tissues that is superior to and safer than TTT or other subthreshold laser treatments described by practitioners of the art.

Microhyperthermal Effect on Laser-Treated Tissues

The normal cellular response to supraphysical levels of heat (hyperthermia) involves a number of coping processes including the mobilization and increased production

(overexpression) of heat shock proteins, which provide valuable cellular survival functions such as protein maintenance, folding, chaperoning and degradation, blocking apoptotic signaling pathways, and, when expressed on the surface of the cell, stabilizing the cell membrane and assisting with cell signaling and receptor function. This activation and overexpression of HSPs is known as the heat shock response. The 70 kDa inducible HSP70 (also known as HSP72) is a key HSP in heat shock response.

The presence of higher concentrations of HSPs inside retinal and optical nerve cells enables them to effectively refold or dispose of abnormal proteins. Aside from the importance of this task in normal cell function, it is a critical task in certain degenerative diseases of the eye; the inability of HSPs to keep up with the accumulation of misfolded protein is a hallmark of the disease. For example, in age-related macular degeneration retinal pigment epithelial (RPE) cells are unable to clear the accumulation of misfolded proteins in lysosomal compartments, leading to the accumulation of lipofuscin in these lysosomes. This places additional stress on the RPE. One of the results is the extracellular accumulation of these lipofuscin-protein aggregates in drusen adjacent to the RPE cells.

HSP70s also serve to block apoptotic signals generated inside stressed or diseased cells. Several key signaling pathways of apoptosis in cells are inhibited by HSP70 (Figure 5). These inhibitory pathways are critical in cells such as retinal neurons. An increase in internal levels of HSP70 increases the ability of stressed neurons to resist apoptosis under conditions of ischemia and hypoxia.

HSP70 also plays an important role in blocking inflammation. One of the key mechanisms in promoting inflammation in tissues is the production and release of the proinflammatory cytokine TNF-a. Expression of TNF-a is mediated by a signal transduction pathway that causes the nuclear translocation of the protein complex nuclear factor kappa- light-chain-enhancer of activated B cells (NF-κβ), which then controls transcription of DNA related to TNF-a. A number of recent publications have linked NF-κβ expression and subsequent release of TNF-a to the destruction of retinal neurons in retinal ischemia (e.g., Lebrun-Julien et al. 2009; Dvoriantchikova et al. 2009). HSP70 blocks this inflammatory pathway by interfering with the NF- κβ essential modulator (NEMO), a key link that prevents the oligomerization of the Ικα- Ικβ kinase complex that leads to NF- κβ nuclear translocation.

The heat shock response has two major triggers. The first trigger is a significant increase in misfolded proteins within the cell that includes the unfolded protein response within the endoplasmic reticulum. Misfolding of nascent and existing proteins can result from heating and is thermal dose dependent. Misfolding and unfolding of protein is induced after cellular temperatures reach 41° C for a sustained period of time. There is a significant temperature inflection point at about 43.5° C above which protein misfolding dramatically increases. The increase in misfolded proteins activates the refolding activity of heat shock proteins inside cells such as HSP27, HSP70, and HSP90. This mobilization also releases normally HSP-bound monomers of heat shock inducing factor 1 (HSF1), a key element to stimulating overexpression of heat shock proteins needed for cellular self-defense against hyperthermia. Under conditions of hyperthermia, HSF1 trimerizes, phosphorylates and translates into the nucleus, where it upregulates genes related to the production of heat shock proteins such as HSP70. HSP70 act as an inhibitor of HSF1 activity through HSF1 monomer binding and blocking of trimer phosphorylation, and increases of cellular HSP70 acts as a homeostatic regulatory mechanism that shuts down the activity of HSF1.

Traditional approaches to stimulating overexpression of heat shock proteins rely on the primary mechanism of thermally-driven protein misfolding. Not surprisingly, the levels of thermal energy that need to be generated in the retinal and optical nerve tissues to obtain

"optimal" HSP70 overexpression are relatively close to or above the levels required for tissue damage. A summary of these studies is included in Table 4. Table 4: Comparison of Optimal Expression Levels of HSP70 and Thermal Damage Thresholds (Minimal Visible Lesion or MVL) with 810 nm Continuous Wave Lasers

1— Desmettre et al. 2001

2— Desmettre et al. 2003

3 -Kim et al. 2006

With the use of longer pulse durations or continuous wave lasers, the dissipation of thermal energy in the tissue is also poorly controlled, leading to inadvertent thermal damage. Even TTT, which is intended to be a subthreshold laser therapy for the retina, is characterized by subclinical thermal damage (e.g., Shields et al. 2002; Morimura et al. 2004). Conventional approaches to laser treatment are not capable of breaking the nexus between HSP70 stimulation and thermal damage to cells in the retina. However, the embodied method of laser treatment can effectively decouple this linkage, inducing significant heat shock protein expression without thermal damage. The invention does this by tapping into two other modes of stimulating HSP overexpression.

The first mode is the stimulation of an alternate pathway to HSF1 activation that utilizes plasma membrane signaling and intracellular calcium generation. A number of studies have shown that the heat shock response can be activated at temperatures below the level of significant protein misfolding. It has now been shown that heat shock response can be directly generated by changes in the morphology of the cellular membrane. The cell membrane is a complex structure of lipids and proteins, but fundamentally comprises a bilipid layer of different constituent phospholipids. This composition is thermosensitive and increases in heat lead to changes in the viscosity and subsequent structural changes within the membrane. At lower temperatures, the membrane exists in a gel state, with tightly packed lipids. At higher temperatures, the membrane achieves a more liquid state, called "hyperfluidity." This change in state is accompanied by conformational changes in the structure of specific phospholipids. Each phospholipid within the membrane has a different temperature at which it makes this transition. Table 5 shows temperature thresholds at whichdifferent lipids in the membrane transition to a hyperfluid state. Table 5: Phase Transition Temperatures for Membrane Phospholipids in Water

Phospholipid Transition Temperature ( Tm), °C

Dipalmitoyl phosphatidic acid (Di 16:0 PA) 67.0

Dipalmitoyl phosphatidylethanolamine (Di 16:0 PE) 63.8

Dipalmitoyl phosphatidylcholine (Di 16:0 PC) 41.4

Dipalmitoyl phosphatidylglycerol (Di 16:0 PG) 41.0

Dilauroyl phosphatidylcholine (Di 14:0 PC) 23.6

Distearoyl phosphatidylcholine (Di 18:0 PC) 58.0

Dioleoyl phosphatidylcholine (Di 18 : 1 PC) -22.0

l-Stearoyl-2-oleoyl-phosphatidylcholine (1-18:0, 2-18: 1 PC) 3.0

Egg phosphatidycholine (Egg PC) -15.0

Adapted from Jain and Wagner 1980; Martonosi 1982

Since the phospholipid composition of the membrane is not homogenous, incremental changes in temperature above 37° C result in gradual changes in the membrane fluid state as well as shifts in the composition of the membrane that include the transition of different types of phospholipids from one bilayer to another. Increased membrane fluidity and changes in phospholipid composition also lead to the aggregation of microdomains within the lipid comprised of phospholipids, cholesterol, and proteins, called lipid rafts. The combination of membrane hyperfluidity and microdomain aggregation, catalyzed by a rise in temperature within the membrane, gives rise to membrane-associated signaling that can by itself induce a heat shock response. One of the key membrane phospholipids mediating low thermal dose heat shock response is phospho lipase C. An established signal transduction pathway that results in the activation of protein kinase C involves G protein coupled receptor (GPCR) signaling through phospho lipase C (Figure 6). In this pathway, a class of GPCRs interact with and activate Gq G-proteins after agonist ligand binding to the GPCR. Activated Gq binds with GTP and, in turn, activates phospholipase C (PLC) to hydrolyze the membrane lipid PIP2, producing IP3 and diacylglycerol. IP3 is water-soluble and diffuses through the cytoplasm to the ER, where it binds to and opens a calcium channel, releasing calcium stores from inside the ER into the cytoplasm.

While the primary mechanism of IP3-induced calcium release in cells is GPCR signaling, it has been established that this pathway can be directly induced by heat- or drug- induced membrane hyperfluidization (Calderwood et al. 1993). Thermal energy therefore is capable of producing sufficient membrane fluidity and driving PLC-mediated calcium release that activates HSF-1. In addition, mild heat shock stress stimulates Racl -mediated activation of HSF1 as well (Han et al. 2001).

Under normal circumstances, heat shock protein activation using this approach requires prolonged heating at gentle temperatures (20 minutes to several hours). However, with a nanosecond laser pulses, high transient temperatures much higher than this can be generated inside the cell. Within a cell where a nanosecond laser pulse is absorbed, temperature gradients may transiently reach temperatures well above even 58° C, the transition temperature for most membrane phospholipids. These transient increases in thermal energy also provide activation energy required to accelerate related signaling pathway. While these phase transitions are ephemeral, lasting less than a microsecond, they are replicated often due to the high frequency repetition rates of the laser. Thus, a situation is created where treated cells repeatedly experience transient high temperature heating capable of activating PLC-mediated calcium release and subsequent HSP70 expression in a much more efficient way than long-pulse or continuous wave laser heating, or simple conduction thermal heating. In addition, transient thermal spikes generated within cells will also induce limited protein misfolding that also catalyzes traditional HSP70 overexpression pathways. However, the overall thermal dose is small enough so that significant misfolding capable of triggering apoptosis does not occur. In addition to increasing the expression of HSP70, it is also likely that these microhyperthermic effects will increase the expression of other heat-induced HSPs such as alpha B-crystallin, HSP27, HSP60, and HSPl 10 that are important to the stabilization and refolding of abnormal proteins and protection of the retinal and optical nerve cells from apoptosis.

Thermoacoustic Effect on Laser Treated Tissues

The heat shock response to subacute microthermal effect is enhanced by the generation of thermoacoustic stress waves in the cellular targets of the laser. It is likely that acoustic stress has a fluidizing effect on cellular membranes independent of thermal stress. Acoustic stress has an additional important effect— the ability to rapidly drive a significant portion of baseline HSP70 out of laser-treated cells. Once liberated from within the cells, these HSPs can function as donor proteins to HSP70-deprived cells and induce generation of additional HSP70.

While all cells have the capacity to overexpress quantities of HSPs in response to stress, there is a baseline level of the different HSPs within cells, including HSP70, necessary to carry out routine functions of protein folding and chaperoning. The baseline level of intracellular HSP70 varies between cell types. In general, cells that play key roles in protecting the body from environmental stress have higher baseline levels of HSP70 under normal conditions. High levels of HSP70 are found in the eye, brain, heart, kidney, and the skin. Specific cell types in these tissues known for high levels of baseline HSP70 include glial cells such as Muller cells, arterial endothelial cells, renal epithelial cells, and

keratinocytes. In addition, cells that subjected to pathogenic stress, such as RPE cells in age- related macular degeneration, also maintain higher levels of HSP70. For the purpose of this discussion, these types of cells will be referred to as "reservoir cells."

It has been shown that the application of heat can lead to release of significant amounts of HSP70 outside of viable cells without inducing apoptosis or necrosis. A number of mechanism for the extracellular release of HSP70 have been proposed, including transport by lipid rafts, small vesicles, or secretory granules, or through the externalization of an endolysosomal compartment. Experiments demonstrating HSP70 release from cells have, for the most part, involved mild heating of cells in a water bath. Released HSP70 under these conditions represented only a portion of the total cell content of HSP70 and occurred over a period of hours from the initial heat shock. The literature describing the results of heating cells with lasers has described resulting HSP overexpression, not extracellular release. An exception to these results involves exposures of skin tissues to a visible light (511/578 nm) from a nanosecond-duration, high pulse frequency laser over a period of one to two minutes, which caused rapid release of HSP70 in a laser dose-dependent manner (Onikienko et al. 2007). Subsequent histology demonstrated that these parameters of laser irradiation induced this effect without causing any damage to the cells within the tissue or disruption of the tissue itself. In a similar fashion it has been shown that nanosecond-duration visible light (532 nm) lasers operating at several kilohertz frequency can also cause the release of HSP70 from skin tissues within the first hour after laser treatment, and that sufficiently high laser doses can mobilize nearly the entire quantity of HSP70 from the reservoir cells in skin. Finally, it has been shown that infra-red lasers can also cause release of HSP70.

The laser-induced rapid release of a significant portion of the baseline HSP70 present in reservoir cells has two results. First, this HSP is available for other cells that have relatively low levels of HSP70, conferring cytoprotection to these cells. This effect has been demonstrated between glial cells and neurons, where HSP70 released by glial cells was taken up by neurons and conferred protection to the neuronal cells (Guzhova et al. 2001). The release of HSP70 from reservoir cells immediately increases cytoprotective status in surrounding cells. Second, the release of HSP70 from reservoir cells also stimulates expression of greater than normal levels of HSP70 in these cells in response to heat stress. This is due to the fact that release of HSP70 decreases the suppressive effects that HSP70 has on HSFl activation. As discussed earlier, overexpression of HSP70 in response to stress is mediated by the HSFl protein. HSFl exists as an inactive monomer in a complex with HSP40/HSP70 and HSP90. Upon stress, such as elevated temperature, HSFl is released from the chaperone complex and trimerizes. HSFl is then transported into the nucleus where it is hyperphosphorylated and binds to DNA containing heat shock elements. The presence of HSP70 inside the cell inhibits the activity of HSFl first by binding with HSFl monomers and subsequently by interfering with HSFl trimer phosphorylation. Because of the suppressive effect of HSP70 on HSFl activation, cells that already possess high baseline levels of HSP70 do not tend to overexpress HSP70 as much as cells with relatively small content of HSP70. By causing the internal store of HSP70 to be depleted, the homeostatic feedback mechanism limiting the HSFl gene is unblocked, allowing the cell to express higher than normal levels of HSP70.

Removal of HSPs from target cells can be accomplished through the use of ultrashort

(nanosecond duration) laser pulses repeated at very high frequencies, preferably in the kilohertz range. While the mechanism of this release is not fully understood, it is known that in response to thermal stress a significant portion of the cytosolic HSP70 translocates to the membrane where it localizes in membrane-associated lysosomes and inserts itself in lipid rafts in association with membrane lipids such as phosphatidylserine. Thermal stress causes transient externalization of phosphatidylserine. It is possible that repeated acoustic pulses, experienced on a cellular scale, triggers release of the HSP70 through exocytosis of these lysosomes and release of lipid rafts.

Thus, the addition of thermoacoustic stress enhances the effect of laser microhyperthermia in a synergistic way, resulting in the rapid mobilization and release of HSP70 from inside of laser treated cells followed by enhanced HSF1 -mediated overexpression of HSP70.

A sustained exposure of tissue to ultrashort pulses at irradiances below the level of microcavitation will result in the transmission of acoustic waves through the tissue, perturbing the plasma and organelle membranes of the cell. This effect is enhanced by the use of high pulse frequencies. Sufficient perturbation of cells leads to biological effects not produced by longer-pulse duration or continuous wave lasers. In particular, coupling hyperthermia with the thermoacoustic effect can induce the rapid release of HSP70 from cells.

By using a laser to create a combination of microhypethermal and thermoacoustic effects in target cells, HSP70 is mobilized in and released from reservoir cells where it is available to HSP70-deprived cells like neurons to aid in cytoprotection. The overall levels of HSP70 in the tissues are increased by microhyperthermically-triggered overexpression. In reservoir cells like glial cells or in stressed RPE cells, the significant and rapid release of HSP70 from the cell disinhibits HSF1 and results in much higher levels of HSP70

overexpression within these cells. These levels of HSP70 have been demonstrated to be neuroprotective and by using this laser approach can be repeatedly triggered in a safe manner. This approach to laser treatment shifts the threshold for optimal HSP70 expression in retinal and optical nerve tissue from about 47° C to below 42° C with equivalent exposure times. It significantly reduces the risk of subclinical damage and makes the chronic use of these types of lasers to treat degenerative retinal and optical nerve disease a realistic approach. The method of the invention utilizes laser irradiation of retinal and optic nerve tissue that generates a photothermal effects and thermoacoustic effects in the treated tissue that is biologically significant and leads to therapeutic benefit while avoiding significant cellular apoptosis or necrosis or any tissue disruption. The method of the invention rapidly releases heat shock proteins from treated tissues and significantly increases expression of HSP70 in these tissues. The method of the invention optimally combines different pulse durations, pulse frequency, pulse energy, wavelength, treatment area, treatment duration and repetition of treatment to produce a therapeutic response without significant damage to or destruction of cells or disruption of tissue. In one embodiment, the pulse durations are between 0.1 and 100 nanoseconds. In another embodiment, the pulse frequency is between 1 and 100 kHz. In another embodiment, the pulse energies are between 1 and 1000 uJ. In yet another embodiment the short rapid pulses are applied to a treatment area between 1-20 mm across and are sustained for 10 to 120 seconds. In another embodiment the treatment is repeated periodically every two days to two weeks to sustain the levels of HSPs within the treated tissues. In yet another embodiment the wavelength used ranges from 750 to 1200 nm.

A practitioner of the art will combine these parameters in a manner appropriate for treatment of a particular disease condition without causing significant damage to or destruction of cells or disruption of tissue. Targeting of different tissues within the eye or different areas of the eye (e.g., macula, fovea, optical disc) will result in the selection of different wavelengths. For example, 810 nm laser light will be more preferably absorbed by melanin-containing RPE cells and choroid cells and by the hemoglobin-containing blood vessels. 1064 nm laser light will be more evenly absorbed by water throughout the tissues. In addition, the transmission of laser energy to the retinal or optical nerve tissues changes with wavelength. Energy transmission efficiencies drops from near 90% at 810 to less than 10% at 1150 nm. The changes in ocular transmission and retinal absorption of laser light is illustrated in Figure 7.

The method of the invention can be used by itself to increase HSP expression and may be used to enhance already elevated levels of HSP. In one embodiment, subjects are first treated with substances that increase the level of HSP70 inside cells of the retina and optic nerve, and are then treated with the laser to force release of this HSP in order to stimulate cells to produce even more HSP70.

Example 1: Prevention of Ischemia-Reperfusion Neuronal Death in the Retina

A number of investigators have linked ischemic injury to the death of retinal neurons. More recently, the mechanism by which neuronal death occurs has been described.

Dvoriantchikova et al. (2009) described that NF-KB-regulated pro-inflammatory and redox- active pathways are central to glial neurotoxicity induced by ischemic injury. Lebrun-Julien et al. (2009) found that nuclear translocation of NF-κΒ in Miiller glial cells led to release of TNF-a, which stimulated increases in surface expression of Ca²⁺-permeable AMPA receptors on neighboring neurons, leading to overexcitation and cell death.

It has already been established that overexpression of HSP70 is neuroprotective in ischemic injury (e.g., Cizkova et al. 2004). HSP70 directly interferes with NF-KB

translocation by dissociating the NF-κβ Essential Modulator (NEMO), preventing

oligomerization of the IKK complex and subsequent NF-κβ activation (Ran et al. 2004; Weiss et al. 2007).

Two key preclinical studies on the protective effect of TTT in acute optic nerve crush injury model have demonstrated both the protective effect of TTT and its ability to increase HSP70 in optical nerve tissues (Kim et al. 2009; Ma et al. 2010). However, both studies utilized thermal doses of laser energy close to the threshold for damage, and in the Ma study laser treatment caused some damage of the optic nerve fibers as well as significant alterations in the chromatin of peripapillary retinal ganglion cells.

In an animal model of retinal ischemia, Norway rats receive a 60-second laser treatment to the right eye. Treatment utilizes a 1064 nm laser operating at 1 nanosecond pulse duration and a 10 kHz pulse frequency. Target spot on the optical disc is 1 mm. An optical lens system ensure that variation of power across the target profile varies by less than 50%. Laser light is delivered to the optical disc using a rodent Goldmann-type fundus contact lens coupled with a microscope slit lamp to eliminate the effects of both corneal refractive power and corneal aberrations. The laser operates at a power level to produce a maximum of 35 W/cm² with peak energies of 350 μ3 at the surface of the treated tissue. Temperature at the surface of the optical disc is measured by thermal imaging to ensure that target temperature (1 mm beneath the surface of the disk) does not exceed 42° C.

Sixty adult female rats are divided into two groups. Group 1 (40) receives laser treatment to the right eye followed by ischemic injury to both eyes. Group 2 (20) receives only laser treatment to the right eye. 24 hours after laser treatment, rats in Group 1 are anesthesized and subject to one hour of ischemia by increasing intraocular pressure to 110 mm Hg in both eyes (following,for example, Stefansson et al. 1988). Subsequent to this procedure, IOP is allowed to return to normal. Twenty of the rats (50 %) in Group 1 and all the rats in Group 2 are sacrificed 24 hours after injury, and the remaining rats in Group 1 are sacrificed seven days after ischemic event. The right and left eyes of all rats are enucleated, fixed and prepared for histology.

Tissue slides from the optical disc are examined for indications of tissue damage. Counts of surviving neurons were made using visual techniques, and determination of apoptosis was done using fluorescent TUNEL techniques. In addition, tissues are analyzed for HSP70 content using standard immunohistochemical staining and Western blotting. The results of the study show that the laser does not result in any damage to neuronal tissues, re-orientation of retinal or optical nerve architecture, cellular alteration or increase in apoptosis over control eyes. Laser treatment of eyes leads to significant increases of HSP70 in the tissues at the time of sacrifice, averaging more than 300% compared to control.

Comparison of rats 24 hours after ischemia showed that laser treated rats had 50% greater levels of HSP70 than control rats. Non-laser treated rats show significant evidence of early apoptosis based on TUNEL compared to laser treated rats.

Comparison of retinal ganglion survival at seven days shows that ischemic injury in the control eyes resulted in a loss of approximately 40% of retinal ganglion cells, compared while less than 20% of the lost retinal ganglion cells within the laser-treated area. Protective effect of the laser did not extend beyond the area of treatment, indicating that cytoprotection was linked to laser treatment.

Example 2: Prevention of Loss of Optic Nerve Function in Rat Model of Glaucoma

Previous studies have demonstrated that the induction of HSP70 can protect retinal ganglion cells in a rat glaucoma model (Park et al. 2001; Qing et al. 2005). To date, no assessment of the effect of nondestructive laser treatment on glaucoma has been assessed in a preclinical model.

In this experiment, 46 adult Wistar rats receive laser photocoagulation of the trabecular meshwork following the combination photocoagulation approach described by Levkovitch-Verbin et al. (2002). Increases in intraocular pressure (IOP) are monitored following the procedure to ensure that IOP increased by at least 100%. Average IOP in glaucomatous rats was 42 mm Hg. Following treatment, 20 rats receive laser

microhyperthermia treatment weekly for eight weeks and 20 rats receive laser

microhyperthermia treatment every other week for eight weeks. Three rats serve as negative controls for IOP, tissue damage and neuronal viability. Three rats serve as positive controls, receiving weekly laser treatment alone but no photocoagulation and provide comparative measurements of tissue damage and HSP70 expression levels. All rats with elevated IOP are treated in the right eye with the left eye serving as the control.

Treatment utilizes a 1064 nm laser operating at 1 nanosecond pulse duration and a 10 kHz pulse frequency. Target spot is centered on the optical disc with a maximum is 3 mm. An optical lens system is coupled with a rodent Goldmann-type fundus contact lens to deliver a uniform level of laser energy to the retina using a microscope slit lamp to eliminate the effects of both corneal refractive power and corneal aberrations. The laser operates at a maximum of 35 W/cm² with peak energies of 350 μ3. Temperature at the surface of the optical disc is measured by thermal imaging to ensure that target temperature (1 mm beneath the surface of the disk) does not exceed 42° C.

Rats are treated for eight weeks and all animals sacrificed. Positive control animals are sacrificed 48 hours after the last laser treatment. One week before sacrifice, all animals with induced glaucoma receive retrograde labeling with Fluoro-Gold. Following sacrifice of the animals, the right and left eyes of all rats are enucleated, fixed and prepared for histology. Cross sections of the optic nerve are also prepared for fluoroscopic examination of retinal ganglion cell density.

Tissue slides from the optical nerve, optical disc and surrounding retina are examined for indications of tissue damage. In addition, tissues are analyzed for HSP70 content using standard immunohistochemical staining and Western blotting.

The results of the study show that the laser does not result in any damage to neuronal tissues, re-orientation of retinal or optical nerve architecture, cellular alteration or increase in apoptosis over control eyes. Laser treatment of eyes leads to significant increases of HSP70 in the tissues at the time of sacrifice, averaging more than 325% compared to controls. Comparison of retinal ganglion survival in glaucomatous rats shows that in glaucomatous control eyes there is a loss of approximately 44% of retinal ganglion cells. In laser treated eyes less than 12% of the retinal ganglion cells within the laser-treated area were lost.

Comparison of HSP70 in glaucomatous control and treated eyes showed that levels of

HSP70 expression in control eyes were only slightly elevated, while in treated eye tissues HSP70 expression amounts were 50%> above normal. Staining showed specific

overexpression in neuronal cells and related glial and astrocytes. Increases in HSP70 expression did not increase at deeper levels in the optic nerve, suggesting that changes in HSP70 expression were due to direct laser treatment, where typical penetration of the laser is 1-2 mm.

Example 3: Treatment of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is characterized by the destruction of the macula of the retina. Wet AMD occurs when abnormal blood vessels behind the retina start to grow under the macula. These new blood vessels tend to be very fragile and often leak blood and fluid. The blood and fluid raise the macula from its normal place at the back of the eye. Damage to the macula occurs rapidly. Dry AMD occurs when the light-sensitive cells in the macula slowly break down. Dry AMD is accompanied by the accumulation of drusens, an extracellular material comprised of yellowish or white aggregates of lipids and proteins.

There are a number of therapeutic approaches to wet AMD including anti-VEGF antibody therapy (Macugen®, Avastin, or Lucentis™) delivered into the eye as well as laser photocoagulation and photodynamic therapy of neovascularizations with or without injection of steroid into the eye. In addition, non-destructive laser therapy in the form of transpupillary thermotherapy (TTT) has been used in an attempt to ameliorate growth of choroidal neovascularization in AMD, but the largest clinical study to date did not show significant efficacy. With respect to dry AMD, which constitute the vast majority of AMD cases, there are no approved drug or device treatments.

A study is done to compare the ability of laser microhyperthermia to provide amelioration of vision loss in dry AMD with geographic atrophy compared with comparable supportive therapy. In this study 40 patients between the ages of 55 and 80 and diagnosed with bilateral geographic atrophy (GA) of dry AMD with lesions < 3 mm in size are enrolled in a randomized study comparing treatment every two weeks with a microhyperthermia laser and a multivitamin supplement regimen consisting of zinc, beta carotene, vitamin C and vitamin E. Subjects in the supplement arm take a daily multivitamin supplement with recommended quantities of minerals and antioxidants based on the current National Eye Institute study.

Subjects in the laser arm receive treatment of active areas of geographic atrophy with an 810 nm laser operating at 10 nanosecond pulse duration and a 10 kHz pulse frequency. An optical lens system is coupled with a microscope slit lamp to deliver a 3 mm spot to the lesion area for 60 seconds. The laser operates at a maximum of 10 W/cm² with peak energies of 100 μΧ Temperature at the surface of the optical disc is measured by thermal imaging to ensure that target temperature at the surface of the lesion does not exceed 42° C.

All subjects are measured for lesion size and visual acuity at the beginning of the study and then quarterly over the next 12 months. Lesion size is assessed using fundus photography. Examination of fundal photography is also used to determine visible damage to the treatment area. Visual acuity is measured using an electronic visual acuity tester.

Subjects in the multivitamin supplementation arm of the study show steady growth in lesion size although average increases in lesion size is less than average for this population group over a one-year period (< 1.8 mm²). Loss of visual acuity (3 lines or greater) occurrs in four patients, with an average loss of acuity of greater than one line. In comparison, no subjects in the microhyperthermia treatment group experience major loss of visual acuity, and on average score 5-7 letters better than control. In the majority of subjects, lesion size stabilizes and in some cases improves, while decrease in drusen is noted in several patients. No patients show any indications of damage or deterioration of vision due to laser treatment.

Example 4: Treatment of Optic Neuritis

Optic neuritis is an inflammation of the optical nerve that can affect the optical disc in the eye (papillitis) as well as the retrobulbar portion. In the West, about one-third of optic neuritis manifests as papillitis, but in Asia it represents two-thirds of cases. Optic neuritis results in degeneration or demyelinization of the nerves. In some cases, such as disease related to multiple sclerosis, attacks can be repeated over time. While individual attacks of optic neuritis do not lead to major loss of visual function, over time there is a loss of visual function associated with the disease. In its typical course, symptoms worsen (including visual loss eye pain) for about two weeks; optic atrophy sets in at about 6-8 weeks and the typical duration of recovery is about 6 months. Standard therapy for this condition is the use of combinations of intravenous and oral steroids. The Optic Neuritis Treatment Trial showed that steroid combinations do not contribute to long-term vision outcome. However, steroids can shorten the duration of the acute incidence.

A study is done to compare the ability of laser microhyperthermia to provide reduction in optical disc inflammation and degeneration, amelioration of vision loss, and reduction in duration of the disease compared with standard IV/oral steroid therapy. In this study 20 patients between the ages of 20 and 40 who have been diagnosed with papillitis with an onset of disease of one week or less than eight days are enrolled in a randomized study comparing treatment of weekly laser microhyperthermia against standard intravenous and oral steroids. Subjects in the steroid arm receive intravenous methylprednisolone 250 mg qid for 3 days with a subsequent oral steroid taper. Subjects in the laser arm receive treatment of the optical disc with a 1064 nm laser operating at 1 nanosecond pulse duration and a 30 kHz pulse frequency. An optical lens system is coupled with a microscope slit lamp to deliver a flat top beam with a spot size of 3 mm in order to cover the optical disc (on average 2.7 mm in diameter). The disk is treated for 120 seconds. The laser operates at a power calculated to deliver a maximum of 20 W/cm² of irradiance to the optical disc surface with peak energies of 47 uJ. Eye tracking technology is utilized to keep the treatment beam on target and temperature at the surface of the optical disc is measured by thermal imaging to ensure that target temperature at the surface of the disk does not exceed 39° C.

Before starting treatment, the optical disc of all subjects are assessed with standard assessments of visual field acuity and contrast sensitivity, funduscopy to establish visual condition of the optical disc and to rule out complications that would represent exclusions from the study, and optical coherence tomography to quantify retinal nerve fiber layer (R FL) thickness.

Subjects are followed in the study for six weeks with testing for visual acuity, fundoscopy and OCT weeks 2 and 6. Laser microhyperthermia patients receive examination before therapeutic laser treatment. Outcomes of the study are resolution of inflammation, nerve fiber thickness, and visual improvements.

Only one subjects in the steroid arm of the study showed normal visual field and contrast sensitivity at two weeks (1 subject with improvement) and 5 subjects do at six weeks. OCT results indicate average RNFL thickness of approximately 94 μΜ at presentation, 84 μΜ at 2 weeks and 68 μΜ at six weeks.

In comparison, subjects in the laser microhyperthermia treatment group show trends toward faster gain in visual acuity. Achievement of normal visual acuity was 2/10 at two weeks and 7/10 by week 6. However, laser-treated subjects show significantly less loss in R FL thickness, with an onset average of approximately 92 μΜ at presentation, 87 μΜ at 2 weeks and 84 μΜ at six weeks. Overall, subjects show trend to faster recovery of visual field acuity without significant thinning of the retinal nerve fiber layer, suggesting that the laser treatment is neuroprotective. In addition, no patients in the laser group show any indications of damage or deterioration of vision due to laser treatment.

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Claims

Claims

1. A method of treating eye diseases of a mammal comprising:

irradiating disease-affected tissues of a retina and/or optical nerve of the mammal with a non-destructive pulsed laser beam,

wherein frequency of the pulses is from about 1 kHz to about 100 kHz,

wherein durations of the pulses is from about 0.1 ns to about 100 ns,

wherein the wavelength of the laser is from about 750 nm to about 1200 nm, wherein the power delivered by the laser beam to the irradiated tissue is from about 1

W/cm² to about 50 W/cm²,

wherein a duration of the irradiation is from about 10 s to about 120 s, and wherein the temperature of the exposed tissue during the irradiation is from about

39°C to about 42°C.

2. The method of Claim 1,

wherein the irradiation is repeated every two days to two weeks.

3. The method of Claim 1 ,

wherein the laser beam is from about 1 mm to about 20 mm across.

4. The method of Claim 1,

wherein the irradiation is delivered directly to the tissue.

5. The method of Claim 1,

wherein the irradiation is delivered through a lens.

6. The method of Claim 1,

wherein the irradiation is delivered through a fiber optic cable.

7. The method of Claim 1,

wherein the irradiation is delivered externally.

8. The method of Claim 1,

wherein the irradiation is delivered internally using additional devices.

9. The method of Claim 1,

wherein the laser beam is produced by a diode laser or a fiber laser.

10. The method of Claim 1,

wherein an intensity profile of the laser beam is parabolic or fiat top.

11. The method of Claim 1 , further comprising

controlling irradiance for tissue pigmentation and temperature during the irradiation.

12. The method of Claim 1,

wherein the diseases are degenerative eye diseases, glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, autosomal dominant drusen, choroidal neovascularization, cystoids macular edema, ischemic retinopathies, Malattia Leventinese, retinal dystrophy, retinitis pigmentosa, retinoschisis, Stargardt's disease, and/or vitelliform macular dystrophy.

13. The method of Claim 1,

wherein the diseases are acute inflammatory diseases of the retina and optic, papillary optical neuritis, neuromyelitis optica, inflammatory optic neuropathy, and/or acute retinitis.

14. The method of Claim 1,

wherein the diseases are acute ischemic conditions of the eye or results of ischemic- reperfusion injury, stroke, or surgery.

15. The method of Claim 1,

wherein the mammal undergoes irradiation before a surgery.

16. The method of Claim 1,

wherein the irradiation is combined with local or systemic administration of a heat shock protein inducing compound.

17. The method of Claim 1,

wherein less than 1% of irradiated cells undergo apoptosis or necrosis.

18. The method of Claim 1,

wherein the power delivered by each pulse of the laser beam is from about 10 uJ to about 1 mJ.

19. The method of Claim 1,

wherein an irradiated area of the tissue is between 1 mm and 20 mm across.

20. A method of releasing heat shock proteins in a mammal comprising:

irradiating retinal and/or optical nerve tissues with a non-destructive pulsed laser beam,

wherein frequency of the pulses is from about 1 kHz to about 100 kHz,

wherein durations of the pulses is from about 0.1 ns to about 100 ns,

wherein the wavelength of the laser is from about 750 nm to about 1200 nm, wherein the power delivered by the laser beam to the irradiated tissue is from about 1

W/cm² to about 50 W/cm²,

wherein a duration of the irradiation is from about 10 s to about 120 s, and wherein the temperature of the exposed tissue during the irradiation is from about

39°C to about 42°C.

21. The method of Claim 20,

wherein the irradiation is repeated every two days to two weeks.

22. The method of Claim 20,

wherein the laser beam is from about 1 mm to about 20 mm across.

23. The method of Claim 20,

wherein the irradiation is delivered directly to the tissue.

24. The method of Claim 20,

wherein the irradiation is delivered through a lens.

25. The method of Claim 20,

wherein the irradiation is delivered through a fiber optic cable.

26. The method of Claim 20,

wherein the irradiation is delivered externally.

27. The method of Claim 20,

wherein the irradiation is delivered internally using additional devices.

28. The method of Claim 20,

wherein the laser beam is produced by a diode laser or a fiber laser.

29. The method of Claim 20,

wherein an intensity profile of the laser beam is parabolic or flat top.

30. The method of Claim 20, further comprising

controlling irradiance for tissue pigmentation and temperature during the irradiation.

31. The method of Claim 20,

wherein the mammal undergoes irradiation before a surgery.

32. The method of Claim 20,

wherein the irradiation is combined with local or systemic administration of a heat shock protein inducing compound.

33. The method of Claim 20,

wherein less than 1% of irradiated cells undergo apoptosis or necrosis.

34. The method of Claim 20,

wherein the power delivered by each pulse of the laser beam is from about 10 uJ to about 1 mJ.

35. The method of Claim 20,

wherein an irradiated area of the tissue is between 1 mm and 20 mm across.

36. The method of Claim 20,

wherein the heat shock proteins comprise HSP 70.

37. A device for treating eye diseases of a mammal comprising:

a laser irradiating disease-affected tissues of a retina and/or optical nerve of the mammal with a non-destructive pulsed laser beam,

wherein frequency of the pulses is from about 1 kHz to about 100 kHz,

wherein durations of the pulses is from about 0.1 ns to about 100 ns,

wherein the wavelength of the laser is from about 750 nm to about 1200 nm, wherein the power delivered by the laser beam to the irradiated tissue is from about 1

W/cm² to about 50 W/cm²,

wherein a duration of the irradiation is from about 10 s to about 120 s, and wherein the temperature of the exposed tissue during the irradiation is from about 39°C to about 42°C.

38. The device of Claim 37,

wherein the irradiation is repeated every two days to two weeks.

39. The device of Claim 37,

wherein the laser beam is from about 1 mm to about 20 mm across.

40. The device of Claim 37,

wherein the irradiation is delivered directly to the tissue.

41. The device of Claim 37,

wherein the irradiation is delivered through a lens.

42. The device of Claim 37,

wherein the irradiation is delivered through a fiber optic cable.

43. The device of Claim 37,

wherein the irradiation is delivered externally.

44. The device of Claim 37,

wherein the irradiation is delivered internally using additional devices.

45. The device of Claim 37,

wherein the laser is a diode laser or a fiber laser.

46. The device of Claim 37,

wherein an intensity profile of the laser beam is parabolic or flat top.

47. The device of Claim 37, further comprising

means for controlling irradiance for tissue pigmentation and temperature during the irradiation.

48. The device of Claim 37,

wherein the diseases are degenerative eye diseases, glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, autosomal dominant drusen, choroidal neovascularization, cystoids macular edema, ischemic retinopathies, Malattia Leventinese, retinal dystrophy, retinitis pigmentosa, retinoschisis, Stargardt's disease, and/or vitelliform macular dystrophy.

49. The device of Claim 37,

wherein the diseases are acute inflammatory diseases of the retina and optic, papillary optical neuritis, neuromyelitis optica, inflammatory optic neuropathy, and/or acute retinitis.

50. The device of Claim 37,

wherein the diseases are acute ischemic conditions of the eye or results of ischemic- reperfusion injury, stroke, or surgery.

51. The device of Claim 37,

wherein the mammal undergoes irradiation before a surgery.

52. The device of Claim 37,

wherein the irradiation is combined with local or systemic administration of a heat shock protein inducing compound.

53. The device of Claim 37,

wherein less than 1% of irradiated cells undergo apoptosis or necrosis.

54. The device of Claim 37,

wherein the power delivered by each pulse of the laser beam is from about 10 uJ to about 1 mJ.

55. The device of Claim 37,

wherein an irradiated area of the tissue is between 1 mm and 20 mm across.

56. A device for releasing heat shock proteins in a mammal comprising:

a laser irradiating retinal and/or optical nerve tissues with a non-destructive pulsed laser beam,

wherein frequency of the pulses is from about 1 kHz to about 100 kHz,

wherein durations of the pulses is from about 0.1 ns to about 100 ns,

wherein the wavelength of the laser is from about 750 nm to about 1200 nm, wherein the power delivered by the laser beam to the irradiated tissue is from about 1

W/cm² to about 50 W/cm²,

wherein a duration of the irradiation is from about 10 s to about 120 s, and wherein the temperature of the exposed tissue during the irradiation is from about

39°C to about 42°C.

57. The device of Claim 56,

wherein the irradiation is repeated every two days to two weeks.

58. The device of Claim 56,

wherein the laser beam is from about 1 mm to about 20 mm across.

59. The device of Claim 56,

wherein the irradiation is delivered directly to the tissue.

60. The device of Claim 56,

wherein the irradiation is delivered through a lens.

61. The device of Claim 56,

wherein the irradiation is delivered through a fiber optic cable.

62. The device of Claim 56,

wherein the irradiation is delivered externally.

63. The device of Claim 56,

wherein the irradiation is delivered internally using additional devices.

64. The device of Claim 56,

wherein the laser is a diode laser or a fiber laser.

65. The device of Claim 56,

wherein an intensity profile of the laser beam is parabolic or flat top.

66. The device of Claim 56, further comprising

means for controlling irradiance for tissue pigmentation and temperature during the irradiation.

67. The device of Claim 56,

wherein the mammal undergoes irradiation before a surgery.

68. The device of Claim 56,

wherein the irradiation is combined with local or systemic administration of a heat shock protein inducing compound.

69. The device of Claim 56,

wherein less than 1% of irradiated cells undergo apoptosis or necrosis.

70. The device of Claim 56,

wherein the power delivered by each pulse of the laser beam is from about 10 uJ to about 1 mJ.

71. The device of Claim 56,

wherein an irradiated area of the tissue is between 1 mm and 20 mm across.

72. The device of Claim 56,

wherein the heat shock proteins comprise HSP 70.