Pre-clinical evaluation of the efficacy and safety of human induced pluripotent stem cell-derived cardiomyocyte patch.

BACKGROUND: Cell- or tissue-based regenerative therapy is an attractive approach to treat heart failure. A tissue patch that can safely and effectively repair damaged heart muscle would greatly improve outcomes for patients with heart failure. In this study, we conducted a preclinical proof-of-concept analysis of the efficacy and safety of clinical-grade human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) patches. METHODS: A clinical-grade hiPSC line was established using peripheral blood mononuclear cells from a healthy volunteer that was homozygous for human leukocyte antigens. The hiPSCs were differentiated into cardiomyocytes. The obtained hiPSC-CMs were cultured on temperature-responsive culture dishes for patch fabrication. The cellular characteristics, safety, and efficacy of hiPSCs, hiPSC-CMs, and hiPSC-CM patches were analyzed. RESULTS: The hiPSC-CMs expressed cardiomyocyte-specific genes and proteins, and electrophysiological analyses revealed that hiPSC-CMs exhibit similar properties to human primary myocardial cells. In vitro and in vivo safety studies indicated that tumorigenic cells were absent. Moreover, whole-genome and exome sequencing revealed no genomic mutations. General toxicity tests also showed no adverse events posttransplantation. A porcine model of myocardial infarction demonstrated significantly improved cardiac function and angiogenesis in response to cytokine secretion from hiPSC-CM patches. No lethal arrhythmias were observed. CONCLUSIONS: hiPSC-CM patches are promising for future translational research and may have clinical application potential for the treatment of heart failure.

Safety confirmation of induced pluripotent stem cell-derived cardiomyocyte patch transplantation for ischemic cardiomyopathy: first three case reports.

Introduction: With the expected increase in patients with heart failure and ischemic 15 cardiomyopathy, the development of myocardial regenerative medicine using cell transplantation as a novel treatment method is progressing. This first-in-human clinical trial aimed to confirm the safety of cardiomyocyte patch transplantation derived from allogeneic induced pluripotent stem (iPS) cells based on the results of several preclinical studies. Study design: The inclusion criteria were left ventricular ejection fraction of 35% or less; heart failure symptoms of New York Heart Association class III or higher despite existing therapies such as revascularization; and a 1-year observation period that included a 3-month immunosuppressive drug administration period after transplantation of iPS cell-derived cardiomyocyte patches to evaluate adverse events, cardiac function, myocardial blood flow, heart failure symptoms, and immune response. Results: In the first three cases of this trial, no transplanted cell-related adverse events were observed during the 1-year observation period, and improvement in heart failure symptoms was observed. In addition, improvements in left ventricular contractility and myocardial blood flow were observed in two of the three patients. Regarding immune response, an increase in transplant cell-specific antibody titer was observed in all three patients after immunosuppressive drug administration. In one patient with poor improvement in cardiac function and myocardial blood flow, an increase in antibody titer against HLA-DQ was observed even before cell transplantation. Conclusions: Our case findings demonstrate that the transplantation of iPS cell-derived cardiomyocyte patches for ischemic cardiomyopathy can be safely performed; however, further investigation of the therapeutic effect and its relationship with an immune response is needed by accumulating the number of patients through continued clinical trials.

Establishment of a protocol to administer immunosuppressive drugs for iPS cell-derived cardiomyocyte patch transplantation in a rat myocardial infarction model.

Transplantation of human allogeneic induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a new, promising treatment for severe heart failure. However, immunorejection is a significant concern in allogeneic hiPSC-CM transplantation, requiring the administration of several immunosuppressive agents. An appropriate protocol for the administration of immunosuppressants may substantially affect the efficacy of hiPSC-CM transplantation in case of heart failure owing to allogeneic transplantation. In this study, we investigated the effect of immunosuppressant administration duration on the efficacy and safety of allogenic hiPSC-CM patch transplantation. We used a rat model of myocardial infarction to evaluate cardiac function using echocardiography six months after the transplantation of hiPSC-CM patches with immunosuppressant administration for either two or four months and compared them to control rats (sham operation, no immunosuppressant administration). Histological analysis performed at 6 months after hiPSC-CM patch transplantation revealed significant improvement in cardiac function in immunosuppressant-treated rats compared with those in the control group. Moreover, fibrosis and cardiomyocyte size was significantly reduced and the number of structurally mature blood vessels was significantly increased in the immunosuppressant-treated rats compared to control rats. However, there were no significant differences between the two immunosuppressant-treated groups. Our results show that prolonged administration of immunosuppressive agents did not enhance the effectiveness of hiPSC-CM patch transplantation, and therefore, highlight the importance of an appropriate immunological regimen for the clinical application of such transplantation.

Case report: Transplantation of human induced pluripotent stem cell-derived cardiomyocyte patches for ischemic cardiomyopathy.

Despite major therapeutic advances, heart failure, as a non-communicable disease, remains a life-threatening disorder, with 26 million patients worldwide, causing more deaths than cancer. Therefore, novel strategies for the treatment of heart failure continue to be an important clinical need. Based on preclinical studies, allogenic human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) patches have been proposed as a potential therapeutic candidate for heart failure. We report the implantation of allogeneic hiPSC-CM patches in a patient with ischemic cardiomyopathy (ClinicalTrials.gov, #jRCT2053190081). The patches were produced under clinical-grade conditions and displayed cardiogenic phenotypes and safety in vivo (severe immunodeficient mice) without any genetic mutations in cancer-related genes. The patches were then implanted via thoracotomy into the left ventricle epicardium of the patient under immunosuppressive agents. Positron emission tomography and computed tomography confirmed the potential efficacy and did not detect tumorigenesis in either the heart or other organs. The clinical symptoms improved 6 months after surgery, without any major adverse events, suggesting that the patches were well-tolerated. Furthermore, changes in the wall motion in the transplanted site were recovered, suggesting a favorable prognosis and the potential tolerance to exercise. This study is the first report of a successful transplant of hiPSC-CMs for severe ischemic cardiomyopathy.

Combined administration of laminin-221 and prostacyclin agonist enhances endogenous cardiac repair in an acute infarct rat heart.

Although endogenous cardiac repair by recruitment of stem cells may serve as a therapeutic approach to healing a damaged heart, how to effectively enhance the migration of stem cells to the damaged heart is unclear. Here, we examined whether the combined administration of prostacyclin agonist (ONO1301), a multiple-cytokine inducer, and stem cell niche laminin-221 (LM221), enhances regeneration through endogenous cardiac repair. We administered ONO1301- and LM221-immersed sheets, LM221-immersed sheets, ONO1301-immersed sheets, and PBS-immersed sheets (control) to an acute infarction rat model. Four weeks later, cardiac function, histology, and cytokine expression were analysed. The combined administration of LM221 and ONO1301 upregulated angiogenic and chemotactic factors in the myocardium after 4 weeks and enhanced the accumulation of ILB4 positive cells, SMA positive cells, and platelet-derived growth factor receptor alpha (PDGFRα) and CD90 double-positive cells, leading to the generation of mature microvascular networks. Interstitial fibrosis reduced and functional recovery was prominent in LM221- and ONO1301-administrated hearts as compared with those in ONO1301-administrated or control hearts. LM221 and ONO1301 combination enhanced recruitment of PDGFRα and CD90 double-positive cells, maturation of vessels, and functional recovery in rat acute myocardial infarction hearts, highlighting a new promising acellular approach for the failed heart.

Elimination of residual undifferentiated induced pluripotent stem cells (iPSCs) using irradiation for safe clinical applications of iPSC-derived cardiomyocytes.

A major concern in the clinical application of induced pluripotent stem cells (iPSCs) is the prevention of tumorigenesis after implantation. Stem cells with high proliferative and differentiation potential are sensitive to radiation. Therefore, we hypothesized that irradiation may selectively eliminate residual undifferentiated human iPSCs (hiPSCs) in a cell population containing differentiated cardiomyocytes derived from hiPSCs (hiPSCs-CMs) and thus reduce tumorigenicity in vivo. hiPSC-CMs were irradiated with X-rays, after which the cell proliferation, apoptosis, morphology, and gene expression were analyzed. The gene expression of Lin28A, Nanog, Oct3/4, and SRY-box 2 was significantly lower in the irradiation group than in the control group. Irradiated hiPSC-CMs showed no change in proliferation potency and morphology compared to untreated hiPSC-CMs. Furthermore, irradiation did not induce apoptosis of differentiated cardiomyocytes. No significant difference in the gene expression of cardiac-specific markers, including α-myosin heavy chain, cardiac troponin T, and NK2 Homeobox 5, was observed between the groups. Tumorigenicity tests using NOG mice showed less frequent tumor formation in the irradiation group than in the control group. Irradiation of hiPSC-CMs significantly reduced the number of undifferentiated hiPSC and the tumor formation, while minimizing any adverse effects on hiPSC-CMs, thereby enabling safe hiPSC-based treatment.

Development of a drug screening system using three-dimensional cardiac tissues containing multiple cell types.

We hypothesized that an appropriate ratio of cardiomyocytes, fibroblasts, endothelial cells, and extracellular matrix (ECM) factors would be required for the development of three-dimensional cardiac tissues (3D-CTs) as drug screening systems. To verify this hypothesis, ECM-coated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), ECM-coated cardiac fibroblasts (CFs), and uncoated cardiac endothelial cells (CEs) were mixed in the following ratios: 10:0:0 (10CT), 7:2:1 (7CT), 5:4:1 (5CT), and 2:7:1 (2CT). The expression of cardiac-, fibroblasts-, and endothelial-specific markers was assessed by FACS, qPCR, and immunostaining while that of ECM-, cell adhesion-, and ion channel-related genes was examined by qPCR. Finally, the contractile properties of the tissues were evaluated in the absence or presence of E-4031 and isoproterenol. The expression of ECM- and adhesion-related genes significantly increased, while that of ion channel-related genes significantly decreased with the CF proportion. Notably, 7CT showed the greatest contractility of all 3D-CTs. When exposed to E-4031 (hERG K channel blocker), 7CT and 5CT showed significantly decreased contractility and increased QT prolongation. Moreover, 10CT and 7CT exhibited a stronger response to isoproterenol than did the other 3D-CTs. Finally, 7CT showed the highest drug sensitivity among all 3D-CTs. In conclusion, 3D-CTs with an appropriate amount of fibroblasts/endothelial cells (7CT in this study) are suitable drug screening systems, e.g. for the detection of drug-induced arrhythmia.

Laminin-511 Supplementation Enhances Stem Cell Localization With Suppression in the Decline of Cardiac Function in Acute Infarct Rats.

BACKGROUND: The extracellular matrix, in particular basement membrane components such as laminins (LMs), is essential for stem cell differentiation and self-renewal. LM511 and LM221 are the main extracellular matrix components of the epicardium, where stem cells were abundant. Here, we examined whether LMs affected the regeneration process by modulating stem cell activities. METHODS: In vitro, adhesive, and proliferative activities of mesenchymal stem cells (MSCs) were evaluated on LM511 and LM221. To examine the effects of LMs in vivo, we established an acute myocardial infarction model by ligation of the proximal part of the left anterior descending artery at the height of the left atrial appendage and then placed atelocollagen sheets with or without LM511 and LM221 over the anterolateral surface of the left ventricular wall. Four or 8 weeks later, cardiac function, histology, and cytokine expressions were analyzed. RESULTS: MSCs showed greater proliferation and adhesive properties on LM511 than on LM221. In vivo, at 4 weeks, isolectin B4-positive cells were significantly higher in the LM511-transplanted group than in the control group. Moreover, some isolectin B4-positive cells expressed both platelet-derived growth factor receptor α and CD90, suggesting that LM511 enhanced MSC recruitment and attachment at the implanted site. After 8 weeks, these cells were more abundant than at 4 weeks. Transplantation with LM511-conjugated sheets increased the expression of cardioprotective and angiogenic factors. CONCLUSIONS: Transplantation with LM511-conjugated sheets enhanced MSC localization to the implantation site and modulated stem cells activities, leading to angiogenesis in acute myocardial infarction rat models.

Tumorigenicity assay essential for facilitating safety studies of hiPSC-derived cardiomyocytes for clinical application.

Transplantation of cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSC-CMs) is a promising treatment for heart failure, but residual undifferentiated hiPSCs and malignant transformed cells may lead to tumor formation. Here we describe a highly sensitive tumorigenicity assay for the detection of these cells in hiPSC-CMs. The soft agar colony formation assay and cell growth analysis were unable to detect malignantly transformed cells in hiPSC-CMs. There were no karyotypic abnormalities during hiPSCs subculture and differentiation. The hiPSC markers TRA1-60 and LIN28 showed the highest sensitivity for detecting undifferentiated hiPSCs among primary cardiomyocytes. Transplantation of hiPSC-CMs with a LIN28-positive fraction > 0.33% resulted in tumor formation in nude rats, whereas no tumors were formed when the fraction was < 0.1%. These findings suggested that combination of these in vitro and in vivo tumorigenecity assays can verify the safety of hiPSC-CMs for cell transplantation therapy.

Immunologic targeting of CD30 eliminates tumourigenic human pluripotent stem cells, allowing safer clinical application of hiPSC-based cell therapy.

Induced pluripotent stem cells (iPSCs) are promising candidate cells for cardiomyogenesis in the failing heart. However, teratoma/tumour formation originating from undifferentiated iPSCs contaminating the graft is a critical concern for clinical application. Here, we hypothesized that brentuximab vedotin, which targets CD30, induces apoptosis in tumourigenic cells, thus increasing the safety of iPSC therapy for heart failure. Flow cytometry analysis identified consistent expression of CD30 in undifferentiated human iPSCs. Addition of brentuximab vedotin in vitro for 72 h efficiently induced cell death in human iPSCs, associated with a significant increase in G2/M phase cells. Brentuximab vedotin significantly reduced Lin28 expression in cardiomyogenically differentiated human iPSCs. Transplantation of human iPSC-derived cardiomyocytes (CMs) without treatment into NOG mice consistently induced teratoma/tumour formation, with a substantial number of Ki-67-positive cells in the graft at 4 months post-transplant, whereas iPSC-derived CMs treated with brentuximab vedotin prior to the transplantation did not show teratoma/tumour formation, which was associated with absence of Ki-67-positive cells in the graft over the same period. These findings suggest that in vitro treatment with brentuximab vedotin, targeting the CD30-positive iPSC fraction, reduced tumourigenicity in human iPSC-derived CMs, potentially providing enhanced safety for iPSC-based cardiomyogenesis therapy in clinical scenarios.

Development of In Vitro Drug-Induced Cardiotoxicity Assay by Using Three-Dimensional Cardiac Tissues Derived from Human Induced Pluripotent Stem Cells.

An in vitro drug-induced cardiotoxicity assay is a critical step in drug discovery for clinical use. The use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is promising for this purpose. However, single hiPSC-CMs are limited in their ability to mimic native cardiac tissue structurally and functionally, and the generation of artificial cardiac tissue using hiPSC-CMs is an ongoing challenging. We therefore developed a new method of constructing three-dimensional (3D) artificial tissues in a short time by coating extracellular matrix (ECM) components on cell surfaces. We hypothesized that 3D cardiac tissues derived from hiPSC-CMs (3D-hiPSC-CT) could be used for an in vitro drug-induced cardiotoxicity assay. 3D-hiPSC-CT were generated by fibronectin and gelatin nanofilm coated single hiPSC-CMs. Histologically, 3D-hiPSC-CT exhibited a sarcomere structure in the myocytes and ECM proteins, such as fibronectin, collagen type I/III, and laminin. The administration of cytotoxic doxorubicin at 5.0 µM induced the release of lactate dehydrogenase, while that at 2.0 µM reduced the cell viability. E-4031, human ether-a-go-go related gene (hERG)-type potassium channel blocker, and isoproterenol induced significant changes both in the Ca transient parameters and contractile parameters in a dose-dependent manner. The 3D-hiPSC-CT exhibited doxorubicin-sensitive cytotoxicity and hERG channel blocker/isoproterenol-sensitive electrical activity in vitro, indicating its usefulness for drug-induced cardiotoxicity assays or drug screening systems for drug discovery.

Building A New Treatment For Heart Failure-Transplantation of Induced Pluripotent Stem Cell-derived Cells into the Heart.

Advanced cardiac failure is a progressive intractable disease and is the main cause of mortality and morbidity worldwide. Since this pathology is represented by a definite decrease in cardiomyocyte number, supplementation of functional cardiomyocytes into the heart would hypothetically be an ideal therapeutic option. Recently, unlimited in vitro production of human functional cardiomyocytes was established by using induced pluripotent stem cell (iPSC) technology, which avoids the use of human embryos. A number of basic studies including ours have shown that transplantation of iPSCderived cardiomyocytes (iPSC-CMs) into the damaged heart leads to recovery of cardiac function, thereby establishing "proof-of-concept" of this iPSC-transplantation therapy. However, considering clinical application of this therapy, its feasibility, safety, and therapeutic efficacy need to be further investigated in the pre-clinical stage. This review summarizes up-to-date important topics related to safety and efficacy of iPSC-CMs transplantation therapy for cardiac disease and discusses the prospects for this treatment in clinical studies.

Presence of trans-synaptic neurons derived from olfactory mucosa transplanted after spinal cord injury.

STUDY DESIGN: Using biotinylated dextran amine (BDA) and wheat germ agglutinin (WGA) tracers, we measured the effectiveness of olfactory mucosa (OM) transplantation as a scaffold in a rat model of chronic spinal cord injury (SCI). OBJECTIVE: We examined whether OM transplantation for chronic SCI in rats results in reconstruction of neuronal pathways by both regeneration of the remaining axons and supply of OM-derived trans-synaptic neurons. SUMMARY OF BACKGROUND DATA: OM is one of the ideal scaffolds for axonal regeneration after chronic SCI. METHODS: Rats received a mild contusion at vertebral level T6-T7. Two weeks after SCI, enhanced green fluorescent protein rat-derived OM, respiratory mucosa, and phosphate-buffered saline were transplanted into each group of SCI rats. Ten weeks after SCI, BDA was injected into the right sensorimotor cortex. Eleven weeks after SCI, WGA was injected into the L1-L2 posterior column to label the corticospinal tract retrogradely and trans-synaptically. Twelve weeks after SCI, rats were killed and their spinal cords were divided into cervical (area a), thoracic-injured (area b), and lower thoracic portions (area c). Immunohistochemically, sections of area (b) were evaluated by counting cells positive for enhanced green fluorescent protein, 4',6-diamidino-2-phenylindole, WGA, and BDA (OM and respiratory mucosa groups). Axonal regenerations were estimated by counting WGA- and BDA-positive dots in transverse sections of area (a) and area (c). RESULTS: Compared with respiratory mucosa and phosphate-buffered saline transplantation, OM transplantation increased the number of WGA-positive dots in area (a), and the number of BDA-positive dots in area (c) was more after OM transplantation than after phosphate-buffered saline transplantation. Furthermore, the number of quadruple-positive cells in area (b) was much higher after OM transplantation. CONCLUSION: Our results provide both indirect and direct evidence for the presence of trans-synaptic neurons. OM transplantation in rats with chronic SCI resulted in reconstruction of neural pathways by both providing trans-synaptic neurons and supporting regeneration of remaining axons. The olfactory mucosa is thought to be an efficacious scaffold to produce the relay neuron in chronic spinal cord injury.

Primary olfactory mucosal cells promote axonal outgrowth in a three-dimensional assay.

Among the possible sources of autologous cells and tissues for use in spinal cord injury grafts, one promising source is the olfactory mucosa containing olfactory ensheathing cells and neural progenitor cells. Olfactory mucosa transplantation for spinal cord injury has been effective in animal models and in pilot clinical trials. However, the contributions of olfactory ensheathing cells and neurons in olfactory mucosa are unclear. For the present study, we prepared primary olfactory mucosal cells and used a cortex-Matrigel coculture assay system to examine the axonal outgrowth of olfactory mucosa. Axonal outgrowth from cortical slices was significantly enhanced in olfactory mucosal cells compared with noncell controls and respiratory mucosal cells, which have few olfactory ensheathing cells and neurons. Axonal outgrowth was severely reduced after treatment with an antineurotrophin cocktail. A conditioned medium in the olfactory mucosa-derived cell group contained neurotrophin-3. Some olfactory ensheathing cells and almost all neurons were immunopositive for neurotrophin-3. Axons originating from cortical slices targeted mainly the astrocyte-like olfactory ensheathing cells. Our findings demonstrate that the axonal outgrowth effect of olfactory mucosa is supported by both olfactory ensheathing cells and neurons in olfactory mucosa.

A new three-dimensional axonal outgrowth assay for central nervous system regeneration.

Although recent studies have shown that cell transplantation is effective in promoting regeneration of the central nervous system (CNS) of adult mammals, functional recovery has been reported to be limited. In vitro models of axonal outgrowth assays are often used as easy methods for screening cells for transplantation but often fail to reflect the physiological conditions of in vivo CNS injury models. In order to bridge the gap between in vitro and in vivo models, we have established a new organotypic co-culture system comprising cortical tissue and a Matrigel containing several cell types that are candidates for transplantation therapy for CNS injury. In this model, cells transplanted in a Matrigel produce a three-dimensional architecture, with axons elongating from the cortex in the Matrigel. The ability of the transplanted cells to promote axonal growth was examined quantitatively by assessing axonal number and length. Moreover, we observed site-specific rearrangement of transplanted cells and interactions between axons and cells, including several cortical cells that migrated into the gel. These results indicate that our co-culture system can provide a useful assay for transplanted cells prior to in vivo screening.

Thiamine tetrahydrofurfuryl disulfide improves energy metabolism and physical performance during physical-fatigue loading in rats.

Impaired energy metabolism is considered a possible cause of fatigue. The thiamine derivative, thiamine tetrahydrofurfuryl disulfide (TTFD), is prescribed and is also an over-the-counter drug for the attenuation of fatigue. It is readily absorbed from the intestinal tract and converted into thiamine pyrophosphate (TPP), which plays an important role as a cofactor for enzymes of metabolic pathways involved in the production of adenosine triphosphate (ATP). We postulated that TTFD has an anti-fatigue effect by improving energy metabolism during physical-fatigue loading. Here, we initially used the forced swimming test to determine whether daily TTFD or thiamine for 5 days has anti-fatigue effects on weight-loaded rats. The swimming duration of TTFD-, but not of thiamine-treated rats, was significantly longer than that of control rats (P < .05). Based on these findings, we examined changes in the levels of thiamine and its phosphate esters in various organs and the effect of TTFD on ATP levels in skeletal muscle after forced swimming, to determine the cellular mechanisms of the anti-fatigue effect of TTFD. Daily TTFD resulted in a characteristic distribution of thiamine and its phosphate esters in rat skeletal muscle, liver, kidney, heart, brain, and plasma. Furthermore, daily TTFD attenuated the decrease in ATP content in the skeletal muscle caused by forced swimming with a weight load for a defined period (150 s). These results indicate that TTFD exerts anti-fatigue effects by improving energy metabolism during physical fatigue.

Targeted tissue oxidation in the cerebral cortex induces local prolonged depolarization and cortical spreading depression in the rat brain.

Spreading depression (SD) has been linked to several neurological disorders as epilepsy, migraine aura, trauma, and cerebral ischemia, which were also influenced by disorderliness of the brain redox homeostasis. To investigate whether local tissue oxidation directly induces SD, we oxidized a restricted local area of the rat cerebral cortex using photo-dynamic tissue oxidation (PDTO) technique and examined the cerebral blood flow (CBF) and direct current (DC) potential in and around the oxidized area. Intensive PDTO induced prolonged depolarization only in the photo-oxidized area, which led to global changes of CBF and DC potential: synchronous negative shifts of DC potential (with an amplitude of approximately 20 mV) and hyperperfusion of CBF occurred. The changes in DC potential and CBF spread at a rate of around 3mm/min beyond the oxidized area to the whole hemisphere of the cerebral cortex, indicating that intensive local oxidation induces SD in the rat brain.

Effects of near-infra-red laser irradiation on adenosine triphosphate and adenosine diphosphate contents of rat brain tissue.

Low-power, near-infra-red laser irradiation has been used to relieve patients from various kinds of pain, though the precise mechanisms of such biological actions of the laser have not yet been resolved. To investigate the cellular mechanisms by near-infra-red laser on the nervous system, we examined the effect of 830-nm laser irradiation on the energy metabolism of the rat brain. The diode laser was applied for 15 min with an irradiance of 4.8 W/cm(2). Tissue adenosine triphosphate (ATP) content of the irradiated area in the cerebral cortex was 19% higher than that of the non-treated area, whereas the adenosine diphosphate (ADP) content showed no significant difference. Laser irradiation at another wavelength (652 nm) had no effect on either ATP or ADP contents. The temperature of the tissue was increased by 4.4-4.7 degrees C during the irradiation of both wavelengths. These results suggest that the increase in tissue ATP content did not result from the thermal effect, but from a specific effect of the laser operated at the 830-nm wavelength.