s2fieldsofstudy: Biology Medicine

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Turning “Cold” Into “Hot” Tumors—Opportunities and Challenges for Radio-Immunotherapy Against Primary and Metastatic Brain Cancers

The development of immunotherapies has revolutionized intervention strategies for a variety of primary cancers. Despite this promising progress, treatment options for primary brain cancer and brain metastasis remain limited and still largely depend on surgical resection, radio- and/or chemotherapy. The paucity in the successful development of immunotherapies for brain cancers can in part be attributed to the traditional view of the brain as an immunologically privileged site. The presence of the blood-brain barrier and the absence of lymphatic drainage were believed to restrict the entry of blood-borne immune and inflammatory cells into the central nervous system (CNS), leading to an exclusion of the brain from systemic immune surveillance. However, recent insight from pre-clinical and clinical studies on the immune landscape of brain cancers challenged this dogma. Recruitment of blood-borne immune cells into the CNS provides unprecedented opportunities for the development of tumor microenvironment (TME)-targeted or immunotherapies against primary and metastatic cancers. Moreover, it is increasingly recognized that in addition to genotoxic effects, ionizing radiation represents a critical modulator of tumor-associated inflammation and synergizes with immunotherapies in adjuvant settings. This review summarizes current knowledge on the cellular and molecular identity of tumor-associated immune cells in primary and metastatic brain cancers and discusses underlying mechanisms by which ionizing radiation modulates the immune response. Detailed mechanistic insight into the effects of radiation on the unique immune landscape of brain cancers is essential for the development of multimodality intervention strategies in which immune-modulatory effects of radiotherapy are exploited to sensitize brain cancers to immunotherapies by converting immunologically “cold” into “hot” environments.

Turning "Cold" Into "Hot" Tumors-Opportunities and Challenges for Radio-Immunotherapy Against Primary and Metastatic Brain Cancers Lisa Sevenich*

Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt, Germany
The development of immunotherapies has revolutionized intervention strategies for a variety of primary cancers. Despite this promising progress, treatment options for primary brain cancer and brain metastasis remain limited and still largely depend on surgical resection, radio-and/or chemotherapy. The paucity in the successful development of immunotherapies for brain cancers can in part be attributed to the traditional view of the brain as an immunologically privileged site. The presence of the blood-brain barrier and the absence of lymphatic drainage were believed to restrict the entry of blood-borne immune and inflammatory cells into the central nervous system (CNS), leading to an exclusion of the brain from systemic immune surveillance. However, recent insight from pre-clinical and clinical studies on the immune landscape of brain cancers challenged this dogma. Recruitment of blood-borne immune cells into the CNS provides unprecedented opportunities for the development of tumor microenvironment (TME)-targeted or immunotherapies against primary and metastatic cancers. Moreover, it is increasingly recognized that in addition to genotoxic effects, ionizing radiation represents a critical modulator of tumor-associated inflammation and synergizes with immunotherapies in adjuvant settings. This review summarizes current knowledge on the cellular and molecular identity of tumor-associated immune cells in primary and metastatic brain cancers and discusses underlying mechanisms by which ionizing radiation modulates the immune response. Detailed mechanistic insight into the effects of radiation on the unique immune landscape of brain cancers is essential for the development of multimodality intervention strategies in which immune-modulatory effects of radiotherapy are exploited to sensitize brain cancers to immunotherapies by converting immunologically "cold" into "hot" environments.

INTRODUCTION
Primary and metastatic brain tumors represent a challenging clinical issue. Glioblastoma (GBM) with an incidence of 2-3 per 100,000 population, is the most common primary brain tumor making up 54% of all gliomas and 16% of all primary brain tumors (1). Brain metastases (BrM), that most frequently arise from melanoma, breast-or lung cancers, are the most common intracranial tumor in adults and exceed the number of primary brain tumors by ∼5-fold (2,3). With the advent of improved control of systemic disease and increased life expectancy of cancer patients, the number of patients with brain metastases is rising (4). The development of cerebral tumors is associated with deteriorated quality of life due to headaches, epileptic seizures, and gradual cognitive impairment (5). Surgical resection, chemo-and radiotherapy (RT) remain the standard of care treatment for patients with brain tumors. Despite recent advances in the development of novel therapies against extracranial tumors, only very little progress has been made in the treatment of cerebral cancers. The majority of clinical trials with immunotherapies for GBM or BrM showed only moderate responses and did not significantly improve progression free survival (PFS) and overall survival (OS) (6). The lack of progress in the development of novel therapies for brain tumors can at least in part be attributed to the unique physiology of the central nervous system (CNS) and in consequence the highly complex brain tumor microenvironment (7). Brain tumors establish an immune suppressive tumor microenvironment that is characterized by high myeloid cell content together with relatively low tumor infiltrating lymphocyte (TIL) numbers and signs of T cell exhaustion (7). While immunotherapy alone fails to provide significant survival benefits for brain cancer patients, there is accumulating evidence, that adjuvant radiotherapy increases tumor immunogenicity and sensitizes brain tumors toward immunotherapy (8,9).
The primary goal of radiotherapy is the induction of DNA damage in rapidly dividing tumor cells to induce different forms of cell death such as apoptosis or mitotic catastrophe (10,11). In contrast to malignantly transformed tumor cells with impaired DNA repair mechanism, non-transformed stromal cells experience less damage given their post-mitotic state and intact DNA repair machinery (10). Although a link between irradiation and the immune system was proposed already 100 years ago (12), anti-tumor effects of radiotherapy were attributed to genotoxic effects on tumor cells, while effects on bystander cells were largely neglected for decades. Radiation dose and fractionation was therefore chosen to induce maximal damage in tumor cells and to spare bystander cells. However, traditional dose regimens might blunt important immune reactions directed against tumors. The discovery of immunogenic cell death (ICD) and abscopal effects provide formal proofs for immunological effects of radiation (13,14). Abscopal effects describe the phenomenon that radiotherapy exerts anti-tumor effects in lesions outside the radiation field by triggering systemic anti-tumor effects (15). Therefore, exploiting the immune modulatory functions of radiotherapy represents an attractive tool to convert immunologically "cold" environments into "hot" environments to increase response rates of immunotherapy. This review will discuss preclinical and clinical evidence that support the applicability of radiotherapy as a sensitizer of immunologically inert tumors, such as GBM and BrM toward immunotherapy with a focus on immune checkpoint blockade (ICB). The field of radio-immunology is just at the beginning to understand the complex cellular and molecular effects of ionizing radiation (IR) on tumor cells and tumor-associated stromal cells that lead to more pronounced and long-lasting immune responses. In addition to clinical observations, it is important to employ preclinical models for systematic evaluation of different treatment regimens in terms of scheduling and dosage to maximize the synergy of radio-immunotherapy. Insight into cellular and molecular effects of radio-immunotherapy is critical to provide a strong scientific rationale for the development of multimodality intervention strategies.
Detailed understanding of the immune landscape of the central nervous system (CNS) at steady state and under pathological conditions is critical to appreciate immunological effects of radiotherapy in brain tumors. This review will therefore first summarize current knowledge on immune surveillance in the CNS and discuss how the development of primary and secondary brain tumors modulates the cellular composition of the TME and alters effector functions of tumor-associated immune cells. Based on this knowledge, different immunological aspects of brain tumors will be discussed to provide insight into the molecular basis of immunotherapy and radiotherapy in combination settings with a particular focus on differences in immune modulation depending on dose and fractionation of IR.

IMMUNE SURVEILLANCE IN THE CENTRAL NERVOUS SYSTEM
The CNS has traditionally been regarded as an immune privileged site that is excluded from systemic immune surveillance (16). Several observations constituted the concept of the immune privileged status of the CNS. First it was noted that the CNS fails to elicit an immune response against immunogenic material that was implanted into the brain parenchyma when avoiding the ventricles and meninges (17,18). Moreover, the presence of the blood-brain-barrier (BBB) or blood-cerebrospinal fluid barrier (BCB) as well as the absence of lymphatic vessels as a route to the lymph node for antigen presenting cells (APC) further underpinned the concept of the CNS immune privilege (16). However, more detailed insights into the anatomical structures of the brain that represent an interface between the CNS and the periphery led to recent revisiting of the immune privilege of the CNS (16,19). The use of single cell sequencing and single cell cytometric approaches helped to elucidate the complexity of immune populations in the steady state CNS (20). In this regard it is important to discriminate between brain regions that are excluded from systemic immune surveillance such as the parenchyma and areas at the border between the CNS and the periphery, including the meninges and the choroid plexus. The presence of the BBB and BCB restrict the entry of immune cell-types as well as the exchange of macromolecules into the brain parenchyma under physiological conditions (16,21). Host defense is therefore performed by microglia, the brain-resident macrophages that constitute the largest population of immune cells in the CNS (22,23) (Figures 1A,B). Parenchymal microglia are long living myeloid cells with self-renewal capacity that arise exclusively from the yolk sac and populate the brain during embryogenesis before the establishment of the BBB (24)(25)(26)(27). As the innate immune cell of the brain, microglia exert key functions in immune surveillance, resolution of infection, wound repair, phagocytosis and debris removal (28). Moreover, microglia are involved in maintaining tissue homeostasis by mediating synaptic pruning, myelo-and neurogenesis as well as neuronal apoptosis (29,30). However, compared to other cells of the macrophage lineage, microglia show lower antigen-presenting capacity (20,31). While the brain parenchyma is tightly shielded from the systemic immune system, there are routes that peripheral leukocytes can utilize to enter the cerebral spinal fluid (CSF), choroid plexus, meninges, and the perivascular space (32). Border-associated myeloid cells (BAMs) including meningeal macrophages (mMF), choroid plexus macrophages (cMF), and perivascular macrophages (pvMF) populate those specialized locations in the CNS (33) (Figures 1A,B). While microglia exclusively originate from yolk sac-derived progenitors, BAM progenitors are of mixed ontological origin deriving from the yolk sac during primitive hematopoiesis and the fetal liver or bone marrow during definitive hematopoiesis (33). Single cell sequencing and mass cytometry (CYTOF) approaches indicate that BAMs show distinct gene expression signatures compared to microglia (20). Importantly, certain subsets of BAMs showed high CD38 and MHCII expression indicating a role in antigen presentation (20). In addition to the tissue-resident macrophages, Ly6Chi and Ly6Clow monocytes as well as dendritic cells constitute the myeloid compartment of the CNS (20,28). Monocytes and DC are primarily localized in the meninges and choroid plexus (34)(35)(36). It was recently demonstrated that myeloid cells, i.e., neutrophils migrate through vascular channels in the skull-dura interface, indicating a direct local interaction between the brain and the skull bone marrow through the meninges as a route for immediate response to brain damage (37).
Moreover, a range of lymphoid cells including B-and T cells as well as innate lymphoid cells (ILC), natural killer (NK), and natural killer T (NKT) cells constitute the lymphoid compartment in the CNS (20). Lymphocytes are absent from the brain parenchyma but can be found within the CSF of the meninges, choroid plexus and the ventricles. Importantly, the recent discovery that the CNS is directly connected to secondary cervical lymph nodes via a standard lymphatic drainage system fundamentally changed the concept of peripheral immune responses within the CNS (38,39). There are three known routes by which intracranial antigens can traffic to CNS draining lymph nodes (40). The first is via ventricular and subarachnoid CSF that is able to cross the cribriform plate and enter the lymphatics draining into the deep cervical lymph nodes (41). Secondly, CSF is able to enter meningeal lymphatics located in the dura that also drain to the deep cervical lymph nodes (39). The third route results from parenchymal interstitial fluid trafficking through the basement membrane of the wall of capillaries and arteries of the brain (42). The first two routes are accessible to immune cells such as T cells, monocytes and DC as well as soluble antigens, while the third route is limited to soluble antigens (40).
The immune landscape in the CNS under steady state has recently been shown to be more complex than previously noted. Based on recent observations it was proposed to refer to the brain as an immunologically distinct rather than privileged site. Despite the description of the cellular constituents of the immune landscape in the CNS, it will be critical to evaluate to which extent the presence of lymphoid and myeloid cells in border-associated areas affects the immune privileged state of the brain parenchyma. In this regard, it is important to identify pathological stimuli that trigger infiltration of immune cells from border-associated areas as a route for immediate response or lead to recruitment of immune cells from the periphery and induction of a systemic response. There is accumulating evidence that different pathological conditions, including neurodegenerative disorders as well as cerebral cancers, induce fundamental changes in the cellular composition of the immune infiltrate and activation state of key players in neuroinflammation (43). Importantly, recent studies revealed that in particular cells that are recruited from the periphery are implicated in the generation of an immune suppressive and cancer permissive environment, while brain-resident cells rather maintain host defense functions (31). This review will focus on tumor-associated inflammation in primary and metastatic brain cancers and highlight similarities and unique characteristics of immune responses that are provoked in the CNS during tumor progression. Understanding the complex immune landscape of brain cancers is critical to develop strategies to overcome the generation of an immune-suppressive environment and perturb traits of tumor cells to escape immune surveillance.

BRAIN TUMORS ESTABLISH AN IMMUNE-SUPPRESSIVE ENVIRONMENT
The development of primary and metastatic brain tumors disrupts the BBB leading to pronounced influx of bloodborne myeloid and lymphoid cells that are usually absent from the brain parenchyma. Tumor-associated macrophages (TAM) represent the most abundant stromal cell type in GBM and BrM often constituting up to 30% of the tumor mass (44,45). Microglia and BMDMs share many phenotypic and functional similarities. The discrimination of both cell types in the context of brain cancers was previously challenging due to their overlapping marker expression and similar morphology in brain tumors. Lineage tracing approaches (46) and the recent discovery of specific markers (31,47,48) significantly contributed to our understanding of cell type specific functions of microglia and BMDMs during disease progression. It was long believed that tumor-associated microglia (TAM-MG) and tumor-associated bone marrow-derived macrophages (TAM-BMDM) exert similar functions in brain tumors. However, gene expression analysis of GBM-associated microglia and macrophages revealed that TAM-MG maintain gene signatures FIGURE 1 | Immune compartments in the CNS. (A) The brain parenchyma is tightly shielded from the systemic immune system by the BBB and BCB that restricts the entry of peripheral immune and inflammatory cells. Yolk sac-derived microglia represent in the innate immune cell type within the brain parenchyma and exert key functions in host defense, immune surveillance and tissue homeostasis. In addition to the brain parenchyma, the CNS comprises structural interfaces that connect the CNS with the periphery, such as the choroid plexus, the meninges and the perivascular spaces. Border-associated macrophages (BAMs) are located in each of these niches. Circulating immune cells such as dendritic cells (DC), B-and T-cells are found in the CSF predominately in the meninges, ventricles and choroid plexus. A meningeal lymphatic system allows DC trafficking from the CNS to CNS draining lymph nodes i.e., cervical lymph nodes. Direct channels that connect the skull bone marrow with the dura enable neutrophils to migrate to the CNS as immediate responders to CNS damage. (B) Relative amount of distinct CNS immune cell populations located in the parenchyma and border areas of the CNS. Depicted amounts are based on data published in Mrdjen et al. (20).
that are associated with house-keeping functions such as synaptic pruning and host defense and induce pro-inflammatory responses. In contrast, recruited TAM-BMDM showed gene signatures that are associated with wound healing, antigen presentation and immune suppression (31,49) (Figure 2). Hence, functional differences of TAMs based on their ontological origin affect their contribution to disease progression and the ratio of TAM-BMDM to TAM-MG is expected to determine prognosis and therapeutic response especially of intervention strategies that aim to reinstate an effective anti-tumor immune response. In addition to evidence from the mouse models, single cell RNAseq analysis confirmed functional differences between TAM-BMDM and TAM-MG based on their ontological origin in human GBM (50). Interestingly, Müller et al. found that TAM-BMDM signatures correlate with significantly shorter survival in low-grade glioma (LGG) with similar trends in GBM, while there is no correlation between survival and TAM-MG signatures (50). Similar to GBM, it was also reported, that TAM-BMDM infiltrate BrM, although to a lesser extent (31). However, it remains unclear if BrM induce similar gene signatures in TAM-MG and TAM-BMDM as described in GBM. In addition to TAM-BMDM, tumor-infiltrating dendritic cells (DC) represent the most important antigen presenting cell type in brain tumors (51). The ability of DC to collect antigens in peripheral organs and to migrate to draining lymph nodes to activate and prime T cells is fundamental for cytotoxic T cell responses directed against specific antigens (52,53). Given the immune privileged status of the brain, it remained unclear whether tumor-specific antigens in the CNS are surveyed by the immune system involving trafficking of DCs from CNS tumors to draining lymph nodes as well as trafficking of primed T cells from cervical lymph nodes into CNS tumors. To address the question on T cell trafficking, Prins et al., performed cell-tracking experiments to follow tumor antigen specific T cells after adoptive transfer of in vitro activated Pmel T cells (54). Imaging of T cell trafficking in this experimental system that is based on systemic vaccination revealed an early accumulation of T cells in all lymphoid organs including the cervical lymph nodes that drain the CNS and a subsequent accumulation in the bone marrow and brain tumors (54). Moreover, Garzon-Muvdi et al. employed an OVAexpressing GBM model with adaptive transfer of OT-1 T cells to identify the site of antigen presentation of tumor-antigens (55). The authors found proliferating OT1 T cells in cervical lymph nodes indicating that antigen presentation and T cell priming against tumor antigens might take place in the CNS draining lymph nodes (Figure 2). T cell expansion in the lymph nodes and anti-tumor effects were most pronounced in response to DC activation by the Toll-like Receptor (TLR)-3 agonist poly(I:C) in combination with PD-1 mediated immunotherapy (55), indicating that a strong proliferative stimulus is needed for effective T cell expansion. Moreover, it remains to be elucidated whether endogenous tumor antigens can elicit T cell priming or if this only occurs in response to highly immunogenic epitopes such as OVA. While the studies by Prins et al. and Garzon-Muvdi et al. provide evidence that under experimental conditions the proposed route of DC migration, T cell priming and local expansion might take place, it is important to acknowledge that the systems are based on strong experimental stimuli that are not expected in naturally grown CNS tumors. Hence, the formal proof of the route in which DCs migrate to cervical lymph nodes to prime T cells that subsequently traffic to CNS tumors to exert cytotoxic functions is still missing to date. While the question on the natural route for DCs and T cells remains to be addressed in CNS tumors, there is strong evidence that activated, cytotoxic T cells that infiltrate CNS tumors encounter a highly immune-suppressive milieu (56)(57)(58)(59). Immune-suppression is particularly well-documented in GBM that are almost completely devoid of T cells (60). Quantitative deficits in the T cell compartment i.e., lymphopenia have been described for GBM patients since the late 1970 (61). It was recently demonstrated that glioblastoma as well as other intracranial tumors induce lymphopenia through sequestration of T cells in the bone marrow leading to a decline in T cell numbers at the tumor site and in lymphoid organs (62). Moreover, T cell apoptosis is induced through interactions of tumor cells via CD70-CD27 signaling (63,64) or through astrocytes-derived FasL (65) at the tumor site. In addition to quantitative effects on T cells, qualitative deficits of T cells are a common phenomenon in patients with intracranial tumors (66). T cell dysfunction in brain tumors can be induced by a variety of mechanisms (67). High levels of immune-suppressive cytokines such as IL6, IL10, and TGFβ dampen T cell proliferation and effector functions (68). Tumor infiltrating lymphocytes (TILs) show high levels of PD-1, CTLA-4, LAG3, TIM3, TIGIT, and CD39 indicating T cell exhaustion (69)(70)(71)(72)(73). Regulatory T cells (Treg) comprise up to 30% of the TILs in GBM that further suppress T cell responses (57,74). Tumor-associated macrophages and microglia have also been shown to support immune suppression and inhibit the expansion of CD4+ and CD8+ T cell expansion while inducing Treg production (31,49,75,76).
The immune landscape of BrM and its consequences on systemic and CNS immunity are less well-characterized compared to primary brain cancers. The question, if immune responses in BrM are predominately driven by the tissue environment or if the cellular identity of the tumor of origin (e.g., melanoma, lung, renal or breast cancer) shapes the mode of inflammation, is currently a field of active research. In contrast to GBM, BrM show moderate or even pronounced T cell infiltration. Several studies reported that the extent and pattern of T cell infiltration depends on the primary tumor entity that metastasizes to the brain (77,78). T cell density is highest in melanoma with a diffuse pattern throughout the metastatic lesion, while renal-, lung-and breast cancer lead to a moderate T cell influx and T cells are dominantly localized within the stromal compartments of the tumor (77). Data from different studies indicate that T cell exhaustion also appears in brain metastasis. For example, expression of PD1 has been found in ∼63% of TILs in melanoma brain metastasis (79,80). A study by Harter et al. demonstrated that high TIL level, PD1+/CD8+ and PDL1 staining were associated with smaller lesions, however there was no significant association with survival (77). In contrast, a study by Berghoff et al., reported a significant correlation of the density of CD3+, CD8+, and CD45RO+ TILs with favorable median survival (78). As observed in GBM, immune suppressive cell types such as Tregs have also been shown to infiltrate experimental models of metastatic melanoma, breast and colon cancer within the brain and were found in patient brain metastases (57,(81)(82)(83)(84).
Taken together, thorough investigation of the tumor microenvironment in GBM and BrM indicate that brain cancers contain the cellular and molecular constituents for therapeutic intervention by checkpoint inhibition. However, clinical data revealed that checkpoint inhibitors as mono-therapy often fail to significantly improve survival rates (85,86). A possible explanation for the inability of checkpoint inhibitors to reinstate an anti-tumor response might be that the majority of infiltrating T cells are bystander cells that are not directed against specific tumor antigens (87). However, it is also possible that T cells with anti-tumor activity are present within brain tumors, but local immune suppression efficiently blunts their cytotoxic activity even in the presence of checkpoint inhibitors (Figure 2). If this is the case, therapeutic strategies that block immune suppression are required to sensitize brain tumors toward immunotherapy. In this context, immunological effects of radiotherapy recently attracted attention and a series of clinical trials have been initiated to test the efficacy of radiotherapy in combination with immunotherapy. The next paragraph will summarize the current status of standard of care and discuss insights from clinical trials with a focus on trials with ICB in GBM and BrM.

Standard of Care
The current standard of care for newly diagnosed GBM is maximal surgical resection with concurrent radiotherapy and temozolomide (TMZ) chemotherapy followed by 6 months of adjuvant TMZ treatment (88). However, despite multimodality therapeutic intervention, GBM has an almost 100% relapse rate with a median time to recurrence of 7 months (89). The clinical situation for patients with recurrent GBM is extremely dire. Surgery is only considered for ∼25% of the patients and reirradiation is only possible as a palliative option in rare cases (90). Moreover, response rates to chemotherapy including TMZ rarely exceed 10% and no effects on OS have been reported (91)(92)(93).
Previous radiation regimen for brain metastasis patients involved whole brain radiotherapy (WBRT) with fractionated doses of 30 Gy in 10 fractions or 20 Gy in 5 fractions. Different clinical trials report a cerebral response following WBRT in 60% of patients and tumor volume reduction after WBRT has been associated with better neurocognitive function and prolonged survival (94). Median survival following WBRT alone in patients with multiple brain metastasis ranges from 3 to 6 months, with 10-15% of patients still alive at 1 year. However, numerous detrimental effects of WBRT in terms of acute and delayed neurotoxicity such as leuko-encephalopathy and loss of memory function as well as radiation necrosis have been described (95,96). Given the lack of survival benefit and both short-and long-term toxicities associated with WBRT, recent guidelines from the European Association of Neuro-Oncology (EANO) recommend a deferment and replacement of WBRT by stereotactic radiosurgery (SRS) for the treatment of patients with a limited number of brain metastases and/or favorable prognostic factors (97). SRS is a single high dose radiation treatment with high accuracy in placing the irradiation field on tumor lesions and improved protection of surrounding tissue. The treatment efficacy of SRS is similar to surgical resection of brain metastases, with local control rates ranging from 80-85% (98). Clinical data also show, that the use of SRS after surgical resection significantly lowers local recurrence compared to surgery alone and that it is associated with a decreased risk of cognitive decline compared to WBRT (99,100). Controversy remains over potential differences of SRS plus WBRT compared to SRS or WBRT alone. A recent meta-analysis that compared the outcome of patients with one or more brain metastases revealed no differences on survival for patients with multiple metastases, while a WBRT plus SRS improved survival in patients with single metastasis. Moreover, WBRT plus SRS resulted in significantly better local tumor control than WBRT alone (101).
Given the dismal prognosis for GBM and BrM patients, in particular patients with recurrent GBM or patients with multiple BrM it is evident that improved intervention strategies are urgently needed to provide better care for brain cancer patients. The introduction of immunotherapy into the clinics for select cancer types led to new hope for an improved management of primary and metastatic brain cancers.

Immunotherapy
Immune checkpoints are an important component of immune responses to keep cytotoxic activity of T cells under control to prevent autoimmunity. Cancers exploit this safety mechanism by up-regulation of checkpoint components on their cell surface to block T cell activity or by co-opting cells of the tumor microenvironment to establish an immune suppressive environment by dampening T cell responses. Checkpoint inhibitors unleash T cells from their inactive or exhausted state to induce anti-tumor responses (102) (Figure 2). To date, the most prominent examples have been antibodies that block the inhibitory immune checkpoint proteins cytotoxic T lymphocyte antigen 4 (CTLA-4), and PD-1 that are expressed predominantly on T cells, or PD-L1 that is expressed on different immune cells as well as aberrantly on tumor cells (103,104). Consequently, successful immunotherapy by checkpoint inhibition relies on the natural ability of T cells to recognize and destroy malignant cells. While GBM and BrM are both characterized by highly immunosuppressive environments, GBM is further characterized by T cell exclusion and low mutational burden resulting in minimal neo-antigen generation (105). In contrast, BrM show moderate to high T cell content depending on the primary tumor entity and the majority of tumors that metastasize to the brain show high mutational load. However, it has to be taken into account that mutations that are found in brain metastasis are often not present in matched primary tumors (106). Data on brain metastasis patients are limited since those patients are often excluded from clinical trials. However, immunotherapies have demonstrated survival benefits for patients with tumors that frequently metastasize to the brain such as melanoma and NSCLC (107,108). The use of checkpoint inhibitors in those patient cohorts allows for retrospective studies to evaluate the efficacy of checkpoint inhibitors against brain metastasis (109), which indicated efficacy of ICB in BrM. Given the potential beneficial effect of ICB in brain metastasis, a limited number of prospective trials have now been initiated to test the efficacy of immunotherapies in the treatment of brain metastases. First clinical trials to evaluate the efficacy of ipilimumab in patients with melanoma brain metastasis reported intracranial responses in 18% of patients with asymptomatic BrM without corticosteroid treatment while only 5% of symptomatic BrM patients on corticosteroid treatment showed intracranial responses (110). This finding further underpins the need that patients are not treated with corticosteroids at the time of ipilimumab treatment. Following clinical trials such as the ABC trial (NCT02374242) (111) and CheckMate-204 (112) aimed to test the efficacy of combining nivolumab and ipilimumab. Both trials report significant intracranial response rates of 46% in the combined treatment group compared to 20% in the nivolumab group in the ABC trial and 57% in the CheckMate-204 trial. Novel combinations are currently explored in clinical trials to further increase the intracranial response and to reduce adverse effects. For example, the activity and safety of the VEGF neutralizing antibody bevacizumab in combination with pembrolizumab or atezolizumab is tested in clinical trial for patients with untreated BrM (NCT02681549; melanoma and NSCLC and NCT03175432 BEAT-MBM; melanoma). Besides the effects of bevacizumab on angiogenesis, there is accumulating evidence, that VEGF blockade leads to reprogramming of the tumor microenvironment from an immunosuppressive to an immune permissive milieu, thus representing a promising combination together with immune checkpoint inhibitors (113). Results from these trials are still pending.
Although multiple factors indicate that GBM harbors intrinsic resistance against checkpoint inhibition as monotherapy, pre-clinical testing showed promising results (114). The CheckMate143 trial (NCT0207717) was the first large-scale randomized clinical trial of PD pathway inhibition in GBM. However, treatment with the PD1 blocking antibody nivolumab failed to extent OS in patients with recurrent GBM, leading to a termination of this trial arm (86). In particular in the context of GBM it is important to take into account that in addition to the immunosuppressive tumor microenvironment, GBM patients often receive TMZ that is known to cause lymphopenia and permanently affect numbers of memory T cells (115). Moreover, corticosteroids such as dexamethasone are commonly used in the treatment of GBM patients to control cerebral edema. However, as already mentioned for BrM patients, corticosteroids are known to adversely affect the efficacy of immunotherapies (116).
In sum, the extent of immune suppression that is established in GBM and BrM together with effects from standard of care treatment that further dampens immune responses might ultimately prevent effective immunotherapies. Therefore, it is important to develop improved intervention strategies that overcome current obstacles to successful immunotherapy in cerebral tumors. In addition to the recently initiated clinical trials that aim to test the efficacy of the combination of different checkpoint inhibitors, there is increasing interest in the potential synergy of radiotherapy and immunotherapy.

Radio-Immunotherapy
Data from retrospective trials suggest that combinations of immunotherapy and radiotherapy significantly increase response rates and show effects on overall survival. For example, Knisely (117). Similarly, ipilimumab plus SRS was shown to increase OS from 5.3 to 18.3 months, while in this study no survival benefits for the combination of ipilimumab plus WBRT was reported (118). Sharverdian et al. reported that within the patient cohort that was enrolled in the KEYNOTE-001 trial (NCT01295827), NSCLC patients that received radiotherapy before pembrolizumab showed better PFS and OS compared to patients who did not receive radiotherapy (119). Ahmed et al. recently reported data from melanoma BrM patients that received nivolumab plus SRS demonstrating high rates of local BrM control of 91% and 85% at the 6 and 12 months follow-up (120). A central question that remains to be addressed in combination trials is the timing of each component for optimal outcome. Possible regimens comprise concurrent, sequential or neo-adjuvant application of the treatment modules (121). So far, the results suggest that the optimal schedule is tumor type and immunotherapy dependent. However, to date, the majority of trials report data that provide evidence for a benefit of concurrent schedules (122) and lowest response rate if radiotherapy is given after the immunotherapy. For example, a study of patients with melanoma brain metastasis showed that concurrent immunotherapy with anti-PD-L1 and anti-CTLA-4 showed improved response rates if immunotherapy was given within a time frame of 4 weeks after radiation compared to treatments that were more than 4 weeks apart (122). Dovedi et al. demonstrated that acquired resistance to fractionated radiation could be overcome by PD-L1 blockade using syngeneic mouse models of melanoma, colorectal and triple-negative breast cancer. However, the effect was only apparent if treatments were applied either concomitantly with or at the end of radiation. The effect was lost if PD-L1 blockade was given 1 week after radiotherapy (123). In contrast, a retrospective analysis of data from 758 patients suggested an improvement of OS with concurrent ICB and RT and hypo-fractionated RT particularly when immune checkpoint inhibition is started at least 1 month before RT, implying a benefit to commence ICB prior to RT (124). To date, pre-clinical and clinical evidence that optimal scheduling of radio-immuno-therapy critically affects the therapeutic response largely stems from studies on extracranial tumors and must be carefully considered for individual cancer types and different checkpoint inhibitors. Defining the optimal schedule for primary and metastatic brain tumors will require carefully designed prospective clinical trials in combination with systematic preclinical testing or mathematical modeling approaches as recently proposed by Serre et al. (125). While there are several ongoing clinical trials that aim to compare the efficacy of immune checkpoint inhibitors in combination with either WBRT or SRS, there are only few trials that are specifically designed to evaluate how different schedules affect safety and efficacy of combined treatment. One phase II clinical trial at the University of Michigan Cancer Center (NCT02097732) that considers the timing of immunotherapy is evaluating the efficacy of an "induction" of ICB prior to SRS [2 doses of ipilimumab prior to SRS, 2 doses of ipilimumab after SRS] vs. "no induction" [SRS first, followed by 4 doses of ipilimumab (3 mg/kg)]. In the future, more clinical trials that address the question on optimal scheduling will be required for conclusive results.
In sum, data from clinical and pre-clinical studies indicate that radiotherapy can act as a sensitizer for immunotherapy. Conventional fractionation takes advantage of the higher radiosensitivity of tumor cells compared to normal cells with respect to DNA repair and cell cycle regulation. However, whether conventional fractionation represents the optimal strategy to maximize synergy with immunotherapy remains unclear to date. Moreover, it will be critical to evaluate whether radiation dose and fractionation that is optimal to induce an immune response in the CNS, is tolerated by the sensitive brain tissue. In order to optimize radiation dose and fractionation as well as scheduling for therapeutic application, it is essential to gain detailed insight into the molecular basis of genotoxic and immune modulatory effects of radiotherapy. The following paragraph will summarize current knowledge on effects of radiotherapy that result from direct damage on tumor cells (i.e., different forms of cell death) with subsequent effects on the inflammatory response against tumors. Moreover, local and systemic immune responses can also be modulated by radiation-induced changes in different cell populations of the tumor microenvironment (Figure 3). Direct and indirect effects on tumor cells and tumor-associated immune cells together determine the extent by which radiotherapy increases immunogenicity of tumors and the synergy between radio-and immunotherapy.

Radiation-Induced Immune Responses Depend on the Type of Cell Death by Which Tumor Cells Are Killed
The central dogma of traditional radiobiology states that effects of radiation on tumor cells are primarily due to the generation of double strand breaks that lead to the induction of different forms of cell death including apoptosis, necrosis, autophagy, or mitotic catastrophe (Figure 3). Apart from the notion that necrosis elicits inflammation due to the release of cellular content, radiotherapy has long been regarded as an immunologically inert process. While immunological effects of radiotherapy were neglected for decades, several discoveries established a link between the immune system and the ability of radiotherapy to achieve tumor control. Stone et al. demonstrated already end of the 70s, that the radiation dose that is required to control tumor growth was twice as high in immune-compromised mice compared to immunecompetent mice (126). Moreover, the occurrence of abscopal effects in which tumor control is achieved in lesions outside the radiation field provides further proof for the contribution of the immune system in tumor control (127). The discovery of immunogenic cell death (ICD) as a molecularly defined processes that leads to priming and activation of immune cells recently led to a paradigm shift. ICD is characterized by the cell surface translocation of calreticulin (CRT), the extracellular release of HMGB1 (High motility group box 1) and of adenosine triphosphate (ATP) (128). Radiotherapy has been shown to induce all three arms of ICD and is therefore regarded as a potent inducer of ICD (129). Different doses or fractionations are believed to induce different forms of cell death (130), and thus modulate downstream cellular responses. Radiation regimens that induce immunologically silent forms of cell death, i.e., apoptotic cell death are therefore not expected to synergize with ICB, while doses and fractionation that trigger inflammatory responses could be used as immune-modulators to induce additive effects of radiotherapy and immunotherapy (131).

Radiation Increases Recognition of Tumor Antigens by the Immune System
Within the process of immune evasion, tumor cells acquire traits that mask the tumor from immune surveillance and destruction. The immune escape stage is characterized by up-regulation of inhibitory ligands and cytokines, reduced MHCI expression, and increased number of suppressive cell types such as Tregs (132). Radiotherapy has been reported to unmask the tumor and thus make it visible again for the innate and adaptive immune system (133). Radiation can up-regulate MHCI expression on the tumor cell surface to enable better antigen presentation of tumor-specific peptides for recognition by cytotoxic T cells (134). Mutational load of tumors is known to correlate with therapeutic response rates to immunotherapy (135)(136)(137)(138). Identification of mutations that drive anti-tumor responses would therefore be of great benefit to identify patients with a high chance of responding to immunotherapy. Moreover, radiation-induced DNA damage can cause an increase in mutational load (139). While an increase in mutational burden might enhance tumor aggressiveness, it might also generate neoantigens that can be recognized and targeted by the immune system (140). Indeed it was demonstrated that IR induces novel peptide synthesis in tumor cells and enhances antigen presentation by MHC class I molecules (134,141). On the other hand, there is evidence that brain tumors show higher systemic tolerance than tumors at extracranial sites. For example, Jackson et al. employed the B16 melanoma model to compare cytotoxic responses against tumors in the CNS and in the periphery. The study showed that CNS melanomas were more tolerogenic than tumors in extracranial sites due to antigen-specific CD8 T cell depletion leading to impaired systemic antitumor immunity (142). The authors concluded, that the observed T cell dysfunction was mainly caused by elevated levels of microgliaderived TGF-β (142). Interestingly, it was demonstrated that the effect of systemic tolerance was reversible by radiotherapy and vaccination.

The cGas-STING Axis in Anti-tumor Immunity
Radiation-induced DNA damage that causes leakage of DNA into the cytosol is known to be sensed by the stimulator of interferon gens (STING) leading to the activation of innate and adaptive immune responses (143). The STING pathway has originally been described as a host defense mechanism to protect organisms against infection with DNA pathogens. When cytosolic DNA is detected, the product of cyclic GMP-AMP synthase (cGAS), cyclic GMP-AMP (cGAMP) activates STING. STING induces the transcription of type I interferon genes via a cascade that involves the STING downstream factors Tank binding kinase (TBK), interferon regulatory factor 3 (IRF3) and nuclear factor kappa light chain enhancer of B cells (NFkB) (144). A growing body of literature suggests that the STING pathway plays a central role in anti-tumor immunity and its expression is lost in several cancer types including colorectal cancer and melanoma (145,146). While several studies linked radiation-induced STING activity to type-I-interferon mediated anti-tumor immunity, there is also evidence that STING activation could drive immunesuppression and radio-resistance via CCR2 mediated recruitment of MDSCs (147). Given the complex cellular and molecular interactions of STING mediated immunological effects, clearly more systemic studies are needed to gain comprehensive mechanistic insight.

Dose and Fractionation Are Critical Parameters for Effective Induction of Immunogenicity
Determining the optimal scheduling for radio-immunotherapy is a major challenge for the field and requires carefully designed prospective clinical studies together with comprehensive studies in animal models to test effects of different treatment regimens (148,149). As discussed above, data from retrospective and prospective clinical trials suggests that treatment schedules in which radiotherapy was given as concurrent, sequential, or neoadjuvant therapy lead to different therapeutic efficacy. Several preclinical studies compared single high-dose with fractionated radiation for their ability to induce immune responses. For example, in a B16-OVA model, both single dose (15 Gy) and fractionated radiation (5x3 Gy) increased the generation of antigen-specific T cells. However, the single 15 Gy dose generated more tumor-infiltrating T cells than conventional fractionation (150). Later it was demonstrated, that the immune response triggered by ablative radiation doses was abrogated by conventional fractionation (151). Moreover, Camphausen et al. demonstrated in a model of Lewis lung carcinoma that 5 × 10 Gy induced more robust abscopal effects than 12 × 2 Gy (152). However, hypo-fractionation might not be favorable when combined with immunotherapy. Dewan et al. demonstrated in a breast cancer model that an abscopal effect was only induced in response to fractionated radiation, not single dose radiation when combined with CTLA-4 inhibition (153). A potential explanation for dose dependent effects of radiation was recently provided by the balance between activation of cGAS-STING signaling vs. Trex1 activation (154). Extremely high single doses (20-30Gy) were shown to blunt immunogenicity by the induction of the DNA exonuclease Trex1. Trex1-mediated degradation of cytosolic DNA consequently abrogates cGAS-STING activation and downstream IFN type1 production. In this study, CTLA-4 blockade did not synergize with high dose irradiation to induce abscopal effects. However, knockdown of Trex1 reinstated synergistic effects of anti-CTLA-4 in combination with high dose radiation (20Gy) (155).

Radiation Modulates the Cellular Composition of the Tumor Microenvironment and Affects Effector Functions of Immune Cells
Another important factor that determines synergy of radioimmunotherapy is the cellular composition of the tumor microenvironment (156). As discussed in the paragraph above, in particular the tumor microenvironment of brain tumors represents a highly complex milieu with brain resident and recruited immune cells (7). Brain tumors are known to establish immune-suppressive environments that are characterized by high myeloid cell content and low percentage of CD8+ effector T cells. Several studies demonstrated that radiotherapy induces increased influx of immune cells into brain tumors. This effect can in part be attributed to effects on the vasculature (157,158). IR also has profound effects on the secretion of cytokines that serve as chemo-attractants for different immune cells including DC and macrophages (159). In addition, IR has been shown to affect key effector functions such as phagocytosis, antigen presentation, and cytotoxicity and alters activation states of immune cells (160)(161)(162). Moreover, radio-sensitivity of T cells has to be taken into account when testing optimal dosage and fractionation. Since immunotherapies rely on functional T cells, their ablation or inactivation is expected to abrogate critical anti-tumor immune responses. Tumor-infiltrating T cells are exposed to radiation and it has been shown that conventional 2 Gy doses given once daily can inactivate T cells (163). This effect is also evidenced by the occurrence of lymphopenia as a common adverse effect associated with whole brain radiotherapy (164) that could significantly dampen anti-tumor immune responses.
Taken together, radiation dose and fractionation have profound effects on the induction of genotoxic and immunogenic effects. Systematic interrogation of the dose dependency of immune responses directed against different cancer types is needed to determine optimal regimens to increase the immunogenicity of tumors and boost the immune system for effective anti-tumor responses that synergize with immunotherapy.

COMBINATION OF IR AND ICB-FUTURE PERSPECTIVES
To date, clinical and pre-clinical data suggest that combining radiotherapy with immunotherapy show higher efficacy compared to mono-therapies. These results represent promising first steps in the quest for improved treatment options for brain cancer patients. However, it is also evident that many hurdles exist that prevent higher response rates and more sustainable anti-tumor reactions. While individual patients show prominent cerebral responses, significant effects on overall survival are rarely reported. This indicates that the pressure of the CNS to establish an immunosuppressive environment is dominant over the attempt to unleash the immune system by immune checkpoint blockade. Based on our current mechanistic understanding of the cellular and molecular drivers of immune-suppression in the steady-state CNS and in the context of cerebral cancers, different approaches appear as viable strategies to overcome the highly immune-suppressive environment in the CNS. Based on the results that TAM-BMDM rather than TAM-MG are implicated in tumor-promotion and immune-suppression, selective depletion or blockade of TAM-BMDM recruitment could lead to more effective T cell activation and execution of anti-tumor effector functions. In addition, systems that would allow more efficient recruitment of T cells into CNS tumors could significantly boost cytotoxic T cell responses directed against tumor neo-antigens. A recent study by Samaha et al. reported the engineering of T cells with an Activated Leukocyte Cell Adhesion Molecule (ALCAM) homing system (HS) (165). In this approach, CD6 (the ligand for ALCAM) was re-engineered to trigger initial anchorage to ALCAM followed by adhesion to ICAM1 expressed on cancer endothelium. Cytotoxic HS T cells infiltrated brain tumors after intravenous injection and showed potent anti-tumor activity. Other strategies that aim to convert immune-suppressive milieus into inflamed environments might utilize neutralizing antibodies against suppressive cytokines such as TGFβ, IL10, or IL6. Moreover, activation of adenosine signaling has been associated with immune-suppression and acquisition of resistance against immunotherapy in different cancer types including melanoma (166)(167)(168)(169). Pharmacological inhibition of enzymes that process ATP into adenosine, i.e., CD39/Entpd1 and CD73/Nt5e or targeting of adenosine receptors are currently evaluated for their potential to block the conversion of a purine-driven, pro-inflammatory environment into an adenosine-driven, immune-suppressive milieu (170)(171)(172)(173). Another promising strategy could employ Trex1 inhibitors to prevent the degradation of cytosolic DNA to more efficiently induce cGAS-STING-IRF signaling to trigger innate immune responses (155). Overcoming the immunesuppressive environment appears to be one of the limiting factors for successful immunotherapy against brain cancers. However, it is also important to keep in mind, that immune suppression is an important safety mechanism that protects the brain from excessive inflammation. Inflammatory responses are often associated with swelling that would harm the delicate structures of the CNS and ultimately lead to brain damage. The increased risk of auto-immunity has been reported in several clinical trials with combination of different ICB. The most recent data on the CheckMate143 trial report that 9 of 10 patients treated with a combination of nivolumab and ipilimumab experienced grade 3 or 4 adverse events with 4 of 10 patients discontinuing therapy due to side effects (83). Therapeutic strategies that aim to convert immune suppressive milieus into inflamed environments should therefore be carefully considered and the potential risk of inducing autoimmunity should be evaluated. Detailed mechanistic insight into pathways that are implicated in cancer-associated immune-suppression and inherent or acquired resistance against brain tumors will hopefully lead to the development of novel multimodality intervention strategies that meet the safety and efficacy criteria for the induction of more efficient and long-lasting anti-tumor immune responses in GBM and BrM patients.

CONCLUDING REMARKS
A close link between radiotherapy and the immune system has been proposed already 100 years ago by Russ and Murphy (12). Murphy's observation that "large doses of x-rays, by destroying the immune conditions, will favor the growth of tumors while small doses, by producing immune conditions will help to overcome the tumor" closely resembles the current view on effects of RT on cancer-associated inflammation (174). Yet, immunogenic effects of radiotherapy have been neglected for decades. The introduction of immunotherapy into the clinic and more detailed molecular insights into the underlying mechanism of immunogenic effects of radiation have recently attracted attention to radiotherapy as a potent modulator of cancer-associated inflammation. While immunotherapy is highly effective for patients with inflamed or hot tumor environments, large patient cohort remain unresponsive to immunotherapy due to intrinsic or acquired resistance. Although we are just at the beginning to understand the cellular and molecular basis that distinguish responders from non-responders, accumulating clinical and pre-clinical data indicate that immunogenic effects of radiotherapy convert cold into hot environments and thus sensitize unresponsive tumors toward immunotherapy. Our current knowledge on radio-immunotherapy against brain cancers is largely based on retrospective clinical trials or preclinical studies on animal models. The initiation of clinical trials and pre-clinical studies that aim to systematically evaluate the effects of different fractionation and treatment regimens is needed to provide deeper insight into optimal schedules to induce synergy between immunotherapy and radiotherapy. While there are certainly indications that favor specific treatment regimens, it is still too early to draw conclusions on effects on PFS or OS. To further improve the response rate, it will be critical to identify molecular pathways that determine the mode of immune responses and to develop strategies that efficiently induce anti-tumor immune responses without the risk of inducing auto-immunity.

AUTHOR CONTRIBUTIONS
The author confirms being the sole contributor of this work and has approved it for publication.


s2fieldsofstudy: Biology Psychology

text:
Transcranial Focused Ultrasound (tFUS) and Transcranial Unfocused Ultrasound (tUS) Neuromodulation: From Theoretical Principles to Stimulation Practices

Transcranial focused ultrasound is an emerging technique for non-invasive neurostimulation. Compared to magnetic or electric non-invasive brain stimulation, this technique has a higher spatial resolution and can reach deep structures. In addition, both animal and human studies suggest that, potentially, different sites of the central and peripheral nervous system can be targeted by this technique. Depending on stimulation parameters, transcranial focused ultrasound is able to determine a wide spectrum of effects, ranging from suppression or facilitation of neural activity to tissue ablation. The aim is to review the state of the art of the human transcranial focused ultrasound neuromodulation literature, including the theoretical principles which underlie the explanation of the bioeffects on neural tissues, and showing the stimulation techniques and parameters used and their outcomes in terms of clinical, neurophysiological or neuroimaging results and safety.

INTRODUCTION
Preliminary animal studies suggest that, potentially, different sites in the peripheral nervous system, from nerves (1) to spinal roots (2), and in the central nervous system, from superficial regions like primary motor cortex (3) or frontal eye field (4), to more deep areas like hippocampus (3), amygdala (5), or thalamus (6) can be targeted by focused ultrasound stimulation technique. In addition, animal studies showed that this technique has a high spatial resolution, useful also for mapping small brain areas, as shown by Fry (7) for the mapping of lateral geniculate nucleus, or by Ballantine et al.
Furthermore, a recent fMRI resting-state functional connectivity animal study (8), showed that the effect of tFUS neuromodulation can last for up to 2 h after stimulation, opening a new way to explore not only the online effect but also the long lasting effect of neuromodulation. The first human transcranial application of ultrasounds for neuromodulation was described by Hameroff et al. (9), with an unfocused transcranial ultrasound (tUS) continuous stimulation of posterior frontal cortex, applied on 31 patients affected by chronic pain. The first human application of focused transcranial ultrasound (tFUS) technique was described by Legon et al. (10). They targeted the primary somatosensory cortex of healthy volunteers, in a within-subjects, sham-controlled study. One of the most interesting results of tFUS applications was a case report of emergence from minimally conscious state, after low intensity non-invasive ultrasonic thalamic stimulation in a patient after acute brain injury (11). Following this first single evidence, a clinical trial is ongoing to explore the effect of thalamic low intensity focused ultrasound in acute brain injury patients (12). Regarding peripheral nervous system neuromodulation, Bailey et al. (13) explored the ability of continuous US at 1. 5 MHz in modulating the ulnar nerve stimulation response to magnetic stimulation (MS). This study showed no significant change in electromyographic response during magnetic plus US ulnar nerve stimulation. However, further studies are needed in order to explore different parameter of stimulation.
In recent years, the scientific community showed a progressive increasing interest on FUS neuromodulation, and some reviews have been published in order to summarize the state of the art on this topic (14)(15)(16)(17)(18).

Mechanisms of Actions of US Neuromodulation
Focused ultrasound is a non-invasive, non-ionizing technique. In order to target a brain region, the first challenge is to let ultrasounds single waves to reach the target at the same time, without different acoustic reflection, refraction, and distortion due to the inhomogeneity of skull bone. This problem can be solved by time shifting each single ultrasound wave, according to the related skull bone acoustical properties, in order to let all the waves to reach the target at the same time (19)(20)(21)(22).
The mechanical interaction between US and neuronal membranes can modify the membrane gating kinetics through the action on mechanosensitive voltage-gated ion channels or neurotransmitter receptors (23)(24)(25). The study of Tyler et al. (25) supports this hypothesis. Their study showed, on ex vivo mouse brains and hippocampal slice cultures, that low-intensity, lowfrequency ultrasound (LILFU) is able to activate voltage-gated sodium and calcium channels. However, this can't be the only mechanism of action, explaining the action potential induction, since in simulations, considering the role of membrane tension on activation of mechanically sensitive voltage gated channels, the resulting effect was too low to induce an excitation (26,27).
In addition, the mechanical action of US is able to induce cavitation into the cellular membrane, by means of membrane pore formation, which changes the membrane permeability. The bilayer sonophore model (28) was introduced to better explain the bioeffects of US, taking into consideration the biomechanical proprieties of US and of cell membranes. According to this model (28), the mechanical energy of US leads to periodic expansions and contractions of the membrane. In this model, the US bioeffect is dependent on the tension applied to the membrane. With a progressive increase in membrane stretch intensity, the bioeffect is mediated by different mechanisms. First by the activation of mechanosensitive proteins. Then, with an increase of intensity, there is a pore formation and with the maximum stretch that can be achieved with the technique a membrane rupture and irreversible lesion is obtained (28) (Figure 1).
Considering the electrical properties of the cell membrane at rest, which can be approximated with a parallel plate capacitor, a hypothesis is that the dynamic fluctuation of the membrane bilayer changes the instantaneous membrane capacitance and leads to a capacitive current, which can potentially activate voltage-dependent sodium and potassium channels (27) (Figure 2). The neuronal bilayer sonophore model (27) combines, in a complementary way, all the biomechanical and bioelectrical proprieties of the cell membrane described, and predicts the stimulation parameter needed to reach a successful motor cortex stimulation. It explains, for example, the higher efficacy of long US stimulation pulses (3, 29,30), and how the action potential can be elicited after the end of the US stimulus (27,31), with a good overlap with the experimental results obtained using real stimulation on the mouse motor cortex (30).

Stimulation Parameters
An acoustic wave can be defined by two fundamental parameters: the intensity, defined as the amplitude of the wave, and the instantaneous period (T), defined as the time needed to complete FIGURE 1 | Ultrasound gradually increases tension in the membrane. From the reference stage (S0), the stretch first activates mechanosensitive proteins (S1); growing tension might damage membrane proteins (S2) and then might induce pore formation (S3a, S3b) or cause membrane rupture [modified, with permission, from Krasovitski et al. (28)]. one single oscillation cycle, which is used to calculate the Acoustic frequency (Af) (Figure 3, Equation 1). In addition to these two parameters, the stimulus duration (StimD) is the total duration of one single sonication.
During the stimulus duration two paradigms of sonication are used: continuous or pulsed. Some of these protocols resemble those used for non-invasive brain stimulation based on repetitive transcranial magnetic stimulation [see Di Lazzaro and Rothwell (32) for a review]. The most used one for neuromodulation is the pulsed paradigm.
For the pulsed paradigm, two additional periods need to be defined: the pulse duration (PD), which is the period of acoustic sonication from the starting point of oscillation to the ending point, before the pause and the pulse repetition period (PRP), which is the period between the starting point of two consecutive sonications, or, in other terms, the sum of the pulse duration (PD) and the pause between two consecutive sonications. This period is used to calculate the pulse repetition frequency (PRF) (Figure 3, Equation 2). For the pulsed paradigm, the duty cycle (DC) (Figure 3, Equation 3) is the fraction of the pulsed repetition period (PRP) covered by the pulse duration (PD). The cycles per pulse (c/p) are the number of cycles during a single pulse (Figure 3, Equation 4); instead, the number of pulses (Np) is the number of pulses throughout the stimulus duration (Figure 3, Equation 5).
The sonication delivered during the stimulus duration period can be repeated, without pauses, for the continuous stimulation protocol. Instead, intermittent protocols are characterized by pauses between the sonications, defined as inter stimulation intervals (ISIs). The intermittent protocol is the most used for FUS neurostimulation, instead the continuous one is the most used for the unfocused stimulation (Table 1).
For safety reasons the indexes that describe the thermal and biomechanical effects of the sonication need to be defined. These parameters are related to the instantaneous intensity of stimulation and its instantaneous acoustic pressure. The two main mechanisms that can induce tissue damage are: local heating, which through proteins denaturation leads to cell death, and inertial cavitation. The latter is thought to be mediated by the collapse of gas bubbles due to the pressure exerted by ultrasonic field sufficiently strong to allow tissue damage.
Both, animal histological studies (8,41,42) and human neuroimaging studies (37,38), showed that it is possible to neuromodulate brain circuits without inducing tissue damage. The thermal index (TI) is the ratio of total acoustic power to the acoustic power required to raise tissue temperature by 1 • C under defined assumptions. Finally, the non-thermal, mechanical bioeffect is described by the mechanical index (MI), which is directly proportional to the ultrasound beam's peak negative pressure and inversely proportional to the frequency of the beam.
The intensity, spatial-peak pulse-average (I SPPA ) is the value of the pulse-average intensity at the point in the acoustic field where the pulse-average intensity is a maximum or is a local maximum within a specified region. The intensity, spatial-peak temporal-average (I SPTA ) is the value of the temporal-average intensity at the point in the acoustic field where the temporalaverage intensity is a maximum, or is a local maximum within a specified region.
The FDA guidelines defined the safety threshold for diagnostic usage of US for adult cephalic ultrasound, which can be applied to neuromodulation. These parameters are Isspa ≤ 190 W/cm 2 , Ispta ≤ 94 mW/cm 2 and a mechanical index ≤ 1.9 (43).

Focused Ultrasound for Targeted Drug Delivery
Focused ultrasound technique can be used also to facilitate drugs delivery in a specific brain area. Until now the most explored application is chemotherapy delivering. However, this versatile technique could be applied for neuromodulation purposes, with different mechanisms.
The first mechanism is a focal blood-brain barrier (BBB) opening, through a transient opening of endothelial tight junctions. Indeed, both animal (44,45) and human (46) studies showed that FUS in combination with microbubbles administered intravenously can open the BBB, in a targeted, noninvasive, safe, and reversible manner. This technique could be used for targeted neuromodulation, with therapy which doesn't cross the BBB. For example Wang et al. (47) showed that it is possible to facilitate gene therapy delivery with recombinant adeno-associated virus, in a non-invasive way, through focused ultrasound targeted BBB opening, with potential applications for optogenetics (48) neuromodulation.
The second system is the local release of drugs, minimizing the effect on other brain areas. Indeed, focused ultrasound can be used to locally release drugs which are administered into the bloodstream through a vehicle (e.g., microbubble, liposome) sensitive to local temperature or pressure changes (49).

METHODS
The literature search methods included the PubMed/MEDLINE databases with the following research string, in Nov 2018: ("Neuromodulation" OR "Brain Stimulation") AND ("focused ultrasound" OR HIFU OR LIFU OR Low-intensity focused ultrasound). After abstract reading and screening, only human studies which described focused ultrasound neuromodulation approaches were included in the present review. In addition to the search protocol described, further articles suggested by experts in the field where read and screened ( Table 1).

Physiological Effects in Normal Subjects
Legon et al. (10) used tFUS to target the human primary somatosensory cortex (S1), showing that tFUS significantly decreased the amplitudes of somatosensory evoked potentials elicited by median nerve stimulation. Furthermore, tFUS significantly modulated the spectral content of sensory-evoked brain oscillations and enhanced the performance on sensory discrimination tasks. The neurophysiologic effects had a spatial resolution of about 1 cm or less.
In another study, tFUS altered EEG intrinsic oscillatory dynamics, preferentially affecting the phase distribution of beta band and modulated the phase rate across beta and gamma frequencies. Furthermore, tFUS affected the phase distributions in the beta band of the early but not of the late components of somatosensory evoked potentials, suggesting a spatial specificity. This hypothesis was supported by the loss of neuromodulatory effects after the displacement of the transducer 1 cm laterally from the original cortical target (39).
Primary (SI) and secondary (SII) somatosensory cortical areas of the hand were targeted in a study by Lee et al. (50), in which two transducers were used. The areas were stimulated separately or simultaneously, under neuronavigation guide. tFUS elicited various types of tactile sensations in the contralateral hand/arm regions. The effects were transient and reversible, and the stimulation resulted safe, as assessed by repeated clinical and neuroradiological evaluations. In addition this study showed, the feasibility of the simultaneous stimulation of different human brain areas. In Lee et al. (38), tFUS stimulation of the human somatosensory cortex elicited somatosensory sensations with anatomical specificity up to a finger, and evoked EEG potentials.
fMRI studies showed the effects of tFUS on cortical and subcortical brain areas, with the ability of high-resolution noninvasive functional brain mapping (33,36,37).
Indeed, Leo et al. (36), demonstrated that tFUS stimulation of cortical (primary motor cortex) and subcortical (head of the caudate) areas can induce blood oxygen level dependent (BOLD) signals in 3T and 7T fMRI, respectively. More recently, pairing tFUS on human primary motor cortex (M1) with 7T BOLD fMRI signals in a cued finger tapping task study, Ai et al. (33) showed that tFUS selectively increases BOLD activation volumes of the target finger representation. These effects did not spatially overcome the sonicated area, and therefore did not involve other motor regions, such as supplementary motor area (SMA) and dorsal premotor cortex (PMd).
tFUS has been used also to target the human primary visual cortex (V1) Lee et al. (37) showed, on BOLD fMRI signals, that tFUS stimulation elicited the activation of a network of brain regions, including V1 and other areas involved in visual and higher-order cognitive processes. Furthermore, stimulation elicited perception of phosphenes and EEG evoked responses.
The effects of tFUS on corticospinal excitability have also been studied through transcranial magnetic stimulation (TMS). Combining a custom-made FUS transducer and a 8-shaped TMS coil, Legon et al. (34) assessed for the first time in humans the effect of concentric and concurrent tFUS/TMS stimulation on M1. The stimulation had an inhibitory effect on single-pulse MEPs and intracortical facilitation, and significantly decreased the reaction time in a motor task. Legon et al. (35) tested the effects tFUS stimulation on sensory thalamus, that was targeted by a single-element focused ultrasound through a neuronavigation system based on the individual subject anatomical MRI. tFUS stimulation inhibited the P14 SEP, and was associated with a change in EEG oscillatory dynamics and to a reduced ability in a tactile judgement task. In addition, this study outlined the value of taking into account the individual skull morphology to produce safe and accurate stimulations.
In a recent single-blind, sham-controlled study (40), tUS was targeted to the motor cortex through a diagnostic imaging ultrasound system. The unfocused stimulation increased MEPs amplitude by 34% compared to baseline, and the increase was recorded up to 6 min after the stimulation. This short-term increase of motor cortex excitability contrasts with a previous findings of MEP inhibition during concurrent tFUS/TMS (34). As discussed by the authors, stimulation parameters and other methodological factors might explain the different findings.
Hameroff et al. (9) used a 8 MHz unfocused transducer to study the effects of transcranial ultrasound stimulation (tUS) on mood, and global affect in 31 patients with chronic pain, in a double-blind, sham-controlled crossover study. Stimulation was targeted to the posterior frontal cortex, contralateral to the most severe pain. After the stimulation, a significant improvement in subjective parameters of global affect derived from the Visual Analog Mood Scale was found.
As part of an ongoing clinical trial on low intensity focused ultrasound in acute brain injury (12), Monti et al. (11) reported a case of emergence from minimally conscious state after low intensity non-invasive ultrasonic thalamic stimulation.

Transcranial Focused vs. Unfocused Ultrasound Neuromodulation
Despite transcranial focused ultrasound (tFUS) and transcranial unfocused ultrasound (tUS) neuromodulation techniques share the same basic mechanisms of action, when applied on the same target they can lead to quite different results.
These results are related to the intrinsic differences between the two techniques. The most important, one is the volume of the brain involved in the ultrasound field. It is intuitive that the volume of the brain involved in the focused or unfocused neuromodulation, and the underlying neural circuits, are crucial to determine the output of the tFUS or tUS neuromodulation. This has been supported also by experimental results, where tFUS and tUS were applied on the same target, the primary motor cortex: tUS increased MEPs amplitude (40) instead tFUS induced a MEP inhibition (34). In addition, the sonication delivered during the stimulus duration period, is generally continuous, without pauses, for tUS, and pulsed, characterized by pauses between the sonications, for tFUS. Low-intensity pulsed FUS is the most effective FUS technique for neuromodulation in both animal model (5, 6) and humans (Table 1). Instead, high intensity continuous FUS is widely used for therapeutic irreversible lesioning (51,55,58,60).

DISCUSSION
Transcranial focused ultrasound is an emerging technique for non-invasive neurostimulation, with direct action on bioelettrical neural activity, and in addition could be used for targeted drug delivery.
Compared to magnetic or electric non-invasive brain stimulation, this technique has a higher spatial resolution and can reach deep structures. In addition, animal studies suggest that, potentially, different sites of the central and peripheral nervous system can be targeted by this technique.
Even if still in a small number, the increasing interest in this technique, led to encouraging results in human studies. These preliminary human studies focused their attention on classic non-invasive neurostimulation targets, like the primary motor cortex, somatosensory area or primary visual cortex, with some studies that explored deep structures like thalamus (11,35) or basal ganglia (36). All showed neurostimulation efficacy in terms of clinical, neurophysiological or functional neuroradiological outcomes ( Table 1).
The data collected since now shows that this technique is safe and well-tolerated, when the stimulation parameters and protocol follow the available guidelines. In addition, tFUS can be also conducted without hair shaving (65). The majority of the studies reported no severe adverse effects. Mild and moderate symptoms are reported such as neck pain, sleepiness, muscle twitches, itchiness, and headache (9, 34) ( Table 1). In future studies, proper assessments, aimed to define the safety parameters for tUS and tFUS, are needed. Finally, every tUS or tFUS protocol should explore the role of auditory confounding factors on the neural responses, in order to show that the effect of stimulation is the consequence only of the targeted area neuromodulation, and not due to an indirect auditory impact (66,67).
Overall, the results up to now encourage the study of tUS and tFUS as non-invasive neuromodulatory techniques in humans. The high spatial resolution of tFUS and the possibility of stimulating cortical and deep brain regions suggest many potential applications, such as cortical and subcortical mapping, the study of functional connectivity, the modulation of neurotransmission. Regarding tUS as a potential neuromodulatory tool, noteworthy is the high accessibility of the devices, which are routinely used in health care settings. Further research is needed to clarify tUS and tFUS efficacy and underlying mechanisms, and to optimize stimulation parameters and targeting accuracy. The initial safety profiles seem promising. A rigorous approach must be maintained in order to ensure safe sonications.


The effects of simvastatin preconditioning on the expression of caspase-3 after myocardial ischemia reperfusion injury in rats

Effect of simvastatin on the expression of caspase-3 in myocardial ischemia reperfusion injury in rats was observed to explore the protective effect of caspase-3 through anti-apoptosis mechanism. A total of 48 healthy male SD rats weighing 160–240 g were selected and divided into 4 groups randomly, namely, the blank group, the sham operation group, the ischemia-reperfusion group and the simvastatin group, with 12 rats in each group. The model of SD rats was made by ligation. The loosen ligature made the reperfusion animal model, the occurrence of arrhythmia in the electrocardiogram of lead II in the experimental animal model was observed, and the area of myocardial infarction in the experimental animal models was detected. The number of apoptotic cells was detected by immunohistochemistry, and the expression of caspase-3 was detected by western blotting. The infarct area in the simvastatin group was significantly lower than the ischemia reperfusion group (P<0.05). The positive rate of the expression of caspase-3 and the positive rate of the expression of apoptotic cells in the ischemic reperfusion and simvastatin groups were significantly higher than that of the blank and sham operation groups, and the positive rate of the expression of caspase-3 and apoptotic cells in the simvastatin group was significantly lower than that of the ischemia-reperfusion group (P<0.05). The arrhythmia score of the simvastatin group was significantly lower than that of the ischemia-reperfusion group (P<0.05). Compared with the blank and sham operation groups, the expression of caspase-3 protein in the ischemia-reperfusion and simvastatin groups was significantly increased, and the expression of caspase-3 protein in the simvastatin group was significantly lower than that of the ischemia reperfusion group (P<0.05). Simvastatin has a protective effect on myocardial ischemia-reperfusion injury, which may be related to the reduction of caspase-3 expression and inhibition of apoptosis.

Introduction
Statins are HMG-CoA reductase inhibitors. There are many statins in the market, such as simvastatin, lovastatin, pravastatin and simvastatin (1). It has been confirmed that statins can make HMG-CoA unable to convert to mevalonic acid which is a necessary substance for the synthesis of cholesterol, which eventually leads to the inhibition of cholesterol synthesis in the body (2). Furthermore, it has been found that after preconditioning with pravastatin, it can effectively inhibit apoptosis of renal ischemia reperfusion injury, and has a better effect on renal protection (3). Statins are also becoming increasingly prominent in the prevention and treatment of heart disease. Necrosis in the ischemic central region is the main manifestation of myocardial ischemia and reperfusion, and there will be obvious apoptosis in the semi-dark zone (4). In this study, an animal model of simvastatin preconditioning myocardial ischemia reperfusion injury is used to observe the effect of the expression of apoptosis inhibitor caspase-3.

Materials and methods
Animals and groups. A total of 48 healthy male SD rats aged 8 weeks and weighing 160-240 g were purchased from Laboratory Animal Center of Sichuan University (Chengdu, China), randomly numbered and divided into 4 groups, namely, the blank group, the sham operation group, the ischemia-reperfusion group and simvastatin group with 12 rats in each group. The feeding environment of the rats was of SPF grade. The ambient temperature was 22±1˚C and the ambient humidity was 45±3%. The rats in each group could eat and drink freely according to the circadian rhythm.
The study was approved by the Ethics Committee of Yancheng TCM Hospital Affiliated to Nanjing University of Chinese Medicine (Yancheng, China). All the experimental operations involved in this experiment were carried out in accordance with the relevant provisions of the NIH guidelines for the use of laboratory animals. Administration. SD rats in the blank group were reared normally. Simvastatin was compounded into suspension state by using medical saline. The amount of 20 mg/kg was administered to SD rats at ten days before operation, once a day at the same time, the same volume of isosmotic saline was used for ten days in the sham operation and ischemia reperfusion groups, once a day at the same time.
Animal model of ischemia-reperfusion injury. According to the method provided by Hadi et al (5), ischemia reperfusion model was established. The 4 groups of SD rats were fasted for 12 h before the model was built. In addition to the blank group, all the SD rats in all the other groups were anesthetized with 10% concentration of chloral hydrate. The limbs of the SD rats were fixed on the operating table. The rat chests were disinfected by medical alcohol after hair removal, the trachea was cut by the surgical blade, using mechanical auxiliary ventilation and the chest was opened along the left edge of the sternum to expose the heart position of the rat. The ligation of the left anterior descending branch of the left coronary artery was covered with the gauze after the wetting of the saline. After half an hour, the ligature was cut through with surgical scissors to restore the blood flow to the reperfusion of the coronary artery. SD rats were sacrificed after 3 h making the model, and the damaged parts of the heart were selected. Formalin solution was used to fixing and paraffin-embedded section was carried out. Then the immunochemistry and TUNEL were performed. Only those with thread without ligation were the sham operation group, and no animals died during the ligation and reperfusion.
Detection of arrhythmia. Referring to the previous scoring methods for ventricular arrhythmias (VA) (6), the score of VE with no VA or <5 times is 0 points, and the score of VE with ≥5 times is 1 point. T at a time <60 sec is 2 points, VT at a time ≥60 sec or multiple accumulation <60 sec is 3 points, and multiple accumulation >60 sec is 4 points. VT occurrence is 5 points, VF occurrence continuously over 5 min or death during the observation period is 6 points.
Detection of myocardial infarction area. The left ventricle was sliced to a thickness of ~2 mm, 5 pieces in total, and put in a 0.25% NBT dyeing solution to stain at 37˚C for 15 min. At the time of necrosis of the myocardium, gray white could be seen, while the non-necrotic myocardium was blue. The water was absorbed with clean absorbent paper, the necrotic area of the myocardium was slowly cut down and weighed. The weight of the necrotic region of the myocardium was recorded, and the proportion of the myocardial weight in the myocardial infarction area accounted for the weight of the left ventricle was calculated.
Immunohistochemical staining. Immunohistochemical examination was performed according to the manufacturer's instructions. Paraffin-embedded treatment was carried out on myocardial tissue block (fixed with 4% polyformaldehyde at 4˚C overnight), paraffin section dewaxing, hydration, PBS washing. After blocking for 15 min, 10% serum at room temperature was blocked in non-specific background for 15 min, anti-caspase-3 monoclonal antibody was added (dilution, 1:200; cat. no. 9662; Cell Signaling Technology, Inc.), and stored in a refrigerator at 4˚C overnight. Rinsing by PBS after taking out the horseradish peroxidase (HRP) labeled secondary antibody (dilution, 1:5,000; cat. no. 3875; Cell Signaling Technology, Inc.) the biotin labeled secondary antibody was added, then incubated at room temperature for 30 min before rinsing. Streptomyces antibiotic protein peroxidase solution was incubated at room temperature for 30 min and then washed again. After DAB staining, rinsing, redying, sealing, and observing under an optical microscope (LSM880; Carl Zeiss AG with Airyscan, Oberkochen, Germany), the cytoplasm of caspase-3 positive cells were brownish or brown color.
Detection of apoptosis by TUNEL. Strictly referring to the operation procedure of one step TUNEL apoptosis in situ assay kits, the tissue block was rinsed with dimethylbenzene and gradient alcohol, and then rinsed and dried after incubating proteinase K at room temperature, and added 50 µl TUNEL liquid, rinsed after incubating in a dark wet box at room temperature for 1 hour, and the 50 µl converted-POD was added, and incubated for another 30 min. After washing, 100 µl DAB substrate was added and incubated for 10 min at room temperature and then rinsed, redyed and dehydrated. After rinsing with xylene, it was sealed and observed under the microscope. Under the optical microscope, it was found that there were brown granules in cytoplasm and nucleus, which was a positive expression. In the positive cell distribution area, 5 different visual fields were randomly selected. The average number was used as the quantitative standard of apoptosis, and the percentage of positive cells was calculated.

Detection of expression of caspase-3 by western blotting.
The protein was extracted according to the steps in the tissue protein extraction instructions. The supernatant was taken as total protein after centrifugation at 12,000 x g for 10 min at 4˚C. The protein was quantified by BCA protein quantitative kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Sampling buffer system with equal concentration was prepared and boiled at 95˚C for 15 min. A total of 10% SDS-PAGE gel was placed and each pore was 15 µl. Then, 100V was used for electrophoresis. After the end of electrophoresis, the protein was transferred to a PVDF membrane by wet method. The membrane was transferred for 2 h (90V), and then it was immersed in the closed liquid, and placed on the rocking bed for 2 h. The caspase-3 (dilution, 1:1,000; cat. no. 9662; Cell Signaling Technology, Inc.) and β-actin primary antibody (dilution, 1:1,000; cat. no. 3700; Cell Signaling Technology, Inc.) was added, then stored in a refrigerator at 4˚C overnight, and β-actin was used as the internal reference. TBST was washed for 10 min at room temperature 3 times. Horseradish enzyme labeled sheep against rabbit IgG antibody (dilution, 1:5,000; cat. no. 3875; Cell Signaling Technology, Inc.) was added and incubated for 1 h at room temperature and then, TBST was washed for 10 min at room temperature 5 times. Fresh ECL luminescent liquid (Cell Signaling Technology Inc.; mixed with liquids A and B) was added to a PVDF film to develop in the dark. After chromogenic photography, the strips obtained were processed and analyzed by ImageJ software (ImageJ2x; Rawak Software, Inc., Dresden, Germany).
Statistical analysis. The SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was used to analyze the data, and the measurement data were expressed as mean ± standard deviation (mean ± SD). Analysis of variance (ANOVA) was used for comparison among groups, and the Bonferroni method was used for comparison between two groups if the variance was homogeneous, while the Welch's method was used for analysis if the variance was not homogeneous. P<0.05 was considered to be statistically different.

Results
Staining diagram of positive apoptosis. Under the optical microscope, it was found that there were brown granules in cytoplasm and nucleus, indicating that the cells in the myocardium were apoptotic (Fig. 1).

Effect of ischemia-reperfusion on infarct area in SD rats.
NBT staining showed that the myocardium of SD rats in the blank and sham operation groups were all stained blue, and no gray white was seen. The myocardium of SD rats in the ischemia reperfusion and simvastatin groups showed a more obvious gray white infarct area which in the simvastatin group was significantly smaller than the ischemia reperfusion group. The difference between the two groups was statistically significant (P=0.007) ( Table I).

Effect of simvastatin on expression of caspase-3 in SD rats.
The expression of caspase-3 in the ischemia-reperfusion group was significantly higher than that in the sham operation group (P<0.001). The expression of caspase-3 in the simvastatin group SD rats was significantly lower than the expression of caspase-3 in the SD rats of the ischemia reperfusion group, and the difference between the two groups was significant (P=0.027) (Fig. 2).    Effect of simvastatin on apoptotic cells in SD rats. Apoptosis was detected in a very small number of cells both in the blank group and the sham operation group. There was a large number of apoptotic cells in the ischemia reperfusion and simvastatin groups, and the number of apoptotic cells in the simvastatin group SD rats was significantly lower than the number of apoptotic cells in the SD rats of the ischemia reperfusion group. The difference between the two groups was statistically significant (P=0.018) (Fig. 3).
Reperfusion arrhythmia monitoring. Arrhythmia in SD rats was not found in the blank and sham operation groups, and the arrhythmia score was 0, while arrhythmia in SD rats was observed in the ischemia reperfusion and simvastatin groups, and the arrhythmia score of the simvastatin group was significantly lower than the arrhythmia score of the ischemia reperfusion group, and the two groups were evaluated. The difference was statistically significant (P=0.033) ( Table II).
Detection of expression of caspase-3 protein by western blotting. Compared with the blank and sham operation groups, the expression of caspase-3 protein in the ischemia-reperfusion and simvastatin groups was significantly higher than the simvastatin group, and the expression of caspase-3 protein in the simvastatin group was significantly lower than the expression of caspase-3 protein in the ischemia-reperfusion group. The difference was statistically significant (P=0.038) (Fig. 4).

Discussion
Four types of cardiac dysfunction occurred after myocardial ischemia reperfusion injury, including reflux, myocardial stunning, fatal reperfusion injury and reperfusion arrhythmia (7,8). At present, there are different explanations for the mechanism of myocardial ischemia-reperfusion injury, such as oxygen free radical theory (9), calcium overload theory (10) and inflammatory response theory (11). Kocak et al (12) suggested that apoptosis would occur during myocardial ischemia-reper--reper-reperfusion injury, and apoptosis plays a decisive role in the final infarct area of myocardium. When a specific drug is used to intervene in the process of apoptosis, the effective inhibition of apoptosis can make the area of the myocardial infarction area up to 70%, significantly improving the state of heart function (13). Therefore, the use of anti-apoptotic drugs in clinical treatment of myocardial ischemia reperfusion injury have a great significance. Apoptosis is also known as programmed cell death (PCD), and caspase-3 is a very important starting factor (14), which mediates PCD process. It has been shown that caspase-3 can be activated by its own cleavage by oligomerization, and can activate a variety of downstream proteases, such as cysteine protease, and participate in the process of inhibiting cell proliferation and inducing cell apoptosis (15). Zheng et al (16) found that caspase-3 inhibitors can effectively inhibit the apoptosis of rabbit cardiopulmonary bypass cardiomyocytes. It is presumed that it may be associated with the inhibition of the activity of caspase-3. Wang et al (17) found that the use of different doses of atorvastatin preconditioning acute myocardial ischemia animal model, can significantly reduce apoptosis of myocardial cells and inhibit the expression of caspase-3 gene. Huang et al (18) found that atorvastatin can inhibit apoptosis of myocardial cells in rats with heart failure after myocardial infarction, and then reduce the structural reconstruction of the heart and improve the cardiac function.
In this study, the expression of caspase-3 was significantly increased in the ischemia-reperfusion and simvastatin groups, which was mainly distributed in the gray white region, indicating that the cardiomyocytes of the SD rats were obviously apoptotic after ischemia-reperfusion, and apoptosis of the simvastatin group was significantly lower than that of the ischemia reperfusion group (P<0.05); the positive rate of caspase-3 was decreased (P<0.05), the area of myocardial infarction area was decreased (P<0.05) and the rate of arrhythmia was significantly decreased (P<0.05). The effect of myocardial apoptosis in the simvastatin group was significantly inhibited, and the incidence of cardiac infarction and arrhythmia was reduced, so as to exert a better protective effect on myocardium, which is in accordance with the results of Ma et al (19). At present, it is not known how simvastatin can protect the myocardium by reducing the expression of caspase-3 through anti-apoptosis. However, Liu et al (20) indicated that the expression of p53 and caspase-3 in simvastatin decreased significantly in the the simvastatin group with simvastatin preconditioning renal ischemia reperfusion, and the level of oxygen free radicals  were also decreased (P<0.05). It is presumed that simvastatin may scavenge the excess oxygen free radicals induced by renal ischemia-reperfusion injury, and then decrease the oxidative damage effect of myocardial cells, reduce the expression of p53 and caspase-3, and ultimately inhibit cell apoptosis. However, for the role of myocardial ischemia reperfusion injury, we need to further design experiments for verification.
In conclusion, simvastatin has a protective effect on myocardial ischemia reperfusion injury, and its mechanism may be associated with reducing the expression of caspase-3 and inhibiting apoptosis.