Cell therapy using ex vivo reprogrammed macrophages enhances antitumor immune responses in melanoma.

BACKGROUND: Macrophage-based cell therapies have shown modest success in clinical trials, which can be attributed to their phenotypic plasticity, where transplanted macrophages get reprogrammed towards a pro-tumor phenotype. In most tumor types, including melanoma, the balance between antitumor M1-like and tumor-promoting M2-like macrophages is critical in defining the local immune response with a higher M1/M2 ratio favoring antitumor immunity. Therefore, designing novel strategies to increase the M1/M2 ratio in the TME has high clinical significance and benefits macrophage-based cell therapies. METHODS: In this study, we reprogrammed antitumor and proinflammatory macrophages ex-vivo with HDAC6 inhibitors (HDAC6i). We administered the reprogrammed macrophages intratumorally as an adoptive cell therapy (ACT) in the syngeneic SM1 murine melanoma model and patient-derived xenograft bearing NSG-SGM3 humanized mouse models. We phenotyped the tumor-infiltrated immune cells by flow cytometry and histological analysis of tumor sections for macrophage markers. We performed bulk RNA-seq profiling of murine bone marrow-derived macrophages treated with vehicle or HDAC6i and single-cell RNA-seq profiling of SM1 tumor-infiltrated immune cells to determine the effect of intratumor macrophage ACT on the tumor microenvironment (TME). We further analyzed the single-cell data to identify key cell-cell interactions and trajectory analysis to determine the fate of tumor-associated macrophages post-ACT. RESULTS: Macrophage ACT resulted in diminished tumor growth in both mouse models. We also demonstrated that HDAC6 inhibition in macrophages suppressed the polarization toward tumor-promoting phenotype by attenuating STAT3-mediated M2 reprogramming. Two weeks post-transplantation, ACT macrophages were viable, and inhibition of HDAC6 rendered intratumor transplanted M1 macrophages resistant to repolarization towards protumor M2 phenotype in-vivo. Further characterization of tumors by flow cytometry, single-cell transcriptomics, and single-cell secretome analyses revealed a significant enrichment of antitumor M1-like macrophages, resulting in increased M1/M2 ratio and infiltration of CD8 effector T-cells. Computational analysis of single-cell RNA-seq data for cell-cell interactions and trajectory analyses indicated activation of monocytes and T-cells in the TME. CONCLUSIONS: In summary, for the first time, we demonstrated the potential of reprogramming macrophages ex-vivo with HDAC6 inhibitors as a viable macrophage cell therapy to treat solid tumors.

Macrophages of multiple hematopoietic origins reside in the developing prostate.

Tissue-resident macrophages contribute to the organogenesis of many tissues. Growth of the prostate is regulated by androgens during puberty, yet androgens are considered immune suppressive. In this study, we characterized the localization, androgen receptor expression and hematopoietic origin of prostate macrophages, and transiently ablated macrophages during postnatal prostate organogenesis in the mouse. We show that myeloid cells were abundant in the prostate during puberty. However, nuclear androgen receptor expression was not detected in most macrophages. We found Cx3cr1, a marker for macrophages, monocytes and dendritic cells, expressed in interstitial macrophages surrounding the prostate and associated with nerve fibers. Furthermore, we provide evidence for the co-existence of embryonic origin, self-renewing, tissue-resident macrophages and recruited macrophages of bone-marrow monocyte origin in the prostate during puberty. Our findings suggest that prostate macrophages promote neural patterning and may shed further light on our understanding of the role of the innate immune system in prostate pathology in response to inflammation and in cancer.

Targeting HDAC6 improves anti-CD47 immunotherapy.

BACKGROUND: Cancer cells can overexpress CD47, an innate immune checkpoint that prevents phagocytosis upon interaction with signal regulatory protein alpha (SIRPα) expressed in macrophages and other myeloid cells. Several clinical trials have reported that CD47 blockade reduces tumor growth in hematological malignancies. However, CD47 blockade has shown modest results in solid tumors, including melanoma. Our group has demonstrated that histone deacetylase 6 inhibitors (HDAC6is) have immunomodulatory properties, such as controlling macrophage phenotype and inflammatory properties. However, the molecular and cellular mechanisms controlling these processes are not fully understood. In this study, we evaluated the role of HDAC6 in regulating the CD47/SIRPα axis and phagocytosis in macrophages. METHODS: We tested the role of HDAC6is, especially Nexturastat A, in regulating macrophage phenotype and phagocytic function using bone marrow-derived macrophages and macrophage cell lines. The modulation of the CD47/SIRPα axis and phagocytosis by HDAC6is was investigated using murine and human melanoma cell lines and macrophages. Phagocytosis was evaluated via coculture assays of macrophages and melanoma cells by flow cytometry and immunofluorescence. Lastly, to evaluate the antitumor activity of Nexturastat A in combination with anti-CD47 or anti-SIRPα antibodies, we performed in vivo studies using the SM1 and/or B16F10 melanoma mouse models. RESULTS: We observed that HDAC6is enhanced the phenotype of antitumoral M1 macrophages while decreasing the protumoral M2 phenotype. In addition, HDAC6 inhibition diminished the expression of SIRPα, increased the expression of other pro-phagocytic signals in macrophages, and downregulated CD47 expression in mouse and human melanoma cells. This regulatory role on the CD47/SIRPα axis translated into enhanced antitumoral phagocytic capacity of macrophages treated with Nexturastat A and anti-CD47. We also observed that the systemic administration of HDAC6i enhanced the in vivo antitumor activity of anti-CD47 blockade in melanoma by modulating macrophage and natural killer cells in the tumor microenvironment. However, Nexturastat A did not enhance the antitumor activity of anti-SIRPα despite its modulation of macrophage populations in the SM1 tumor microenvironment. CONCLUSIONS: Our results demonstrate the critical regulatory role of HDAC6 in phagocytosis and innate immunity for the first time, further underscoring the use of these inhibitors to potentiate CD47 immune checkpoint blockade therapeutic strategies.

TRIM28 promotes luminal cell plasticity in a mouse model of prostate cancer.

The Tripartite motif-containing 28 (TRIM28) transcriptional cofactor is significantly upregulated in high-grade and metastatic prostate cancers. To study the role of TRIM28 in prostate cancer progression in vivo, we generated a genetically-engineered mouse model, combining prostate-specific inactivation of Trp53, Pten and Trim28. Trim28 inactivated NPp53T mice developed an inflammatory response and necrosis in prostate lumens. By conducting single-cell RNA sequencing, we found that NPp53T prostates had fewer luminal cells resembling proximal luminal lineage cells, which are cells with progenitor activity enriched in proximal prostates and prostate invagination tips in wild-type mice with analogous populations in human prostates. However, despite increased apoptosis and reduction of cells expressing proximal luminal cell markers, we found that NPp53T mouse prostates evolved and progressed to invasive prostate carcinoma with a shortened overall survival. Altogether, our findings suggest that TRIM28 promotes expression of proximal luminal cell markers in prostate tumor cells and provides insights into TRIM28 function in prostate tumor plasticity.

OncoLoop: A Network-Based Precision Cancer Medicine Framework.

Prioritizing treatments for individual patients with cancer remains challenging, and performing coclinical studies using patient-derived models in real time is often unfeasible. To circumvent these challenges, we introduce OncoLoop, a precision medicine framework that predicts drug sensitivity in human tumors and their preexisting high-fidelity (cognate) model(s) by leveraging drug perturbation profiles. As a proof of concept, we applied OncoLoop to prostate cancer using genetically engineered mouse models (GEMM) that recapitulate a broad spectrum of disease states, including castration-resistant, metastatic, and neuroendocrine prostate cancer. Interrogation of human prostate cancer cohorts by Master Regulator (MR) conservation analysis revealed that most patients with advanced prostate cancer were represented by at least one cognate GEMM-derived tumor (GEMM-DT). Drugs predicted to invert MR activity in patients and their cognate GEMM-DTs were successfully validated in allograft, syngeneic, and patient-derived xenograft (PDX) models of tumors and metastasis. Furthermore, OncoLoop-predicted drugs enhanced the efficacy of clinically relevant drugs, namely, the PD-1 inhibitor nivolumab and the AR inhibitor enzalutamide. SIGNIFICANCE: OncoLoop is a transcriptomic-based experimental and computational framework that can support rapid-turnaround coclinical studies to identify and validate drugs for individual patients, which can then be readily adapted to clinical practice. This framework should be applicable in many cancer contexts for which appropriate models and drug perturbation data are available. This article is highlighted in the In This Issue feature, p. 247.

Prostate organogenesis.

Prostate organogenesis begins during embryonic development and continues through puberty when the prostate becomes an important exocrine gland of the male reproductive system. The specification and growth of the prostate is regulated by androgens and is largely a result of cell-cell communication between the epithelium and mesenchyme. The fields of developmental and cancer biology have long been interested in prostate organogenesis because of its relevance for understanding prostate diseases, and research has expanded in recent years with the advent of novel technologies, including genetic-lineage tracing, single-cell RNA sequencing and organoid culture methods, that have provided important insights into androgen regulation, epithelial cell origins and cellular heterogeneity. We discuss these findings, putting them into context with what is currently known about prostate organogenesis.

Mantle cell lymphoma polarizes tumor-associated macrophages into M2-like macrophages, which in turn promote tumorigenesis.

Tumor-associated macrophages (TAMs) are recognized as a hallmark of certain solid cancers and predictors of poor prognosis; however, the functional role of TAMs in lymphoid malignancies, including B-cell lymphoma, has not been well defined. We identified infiltration of F4/80+ TAMs in a syngeneic mouse model using the recently generated murine mantle cell lymphoma (MCL) cell line FC-muMCL1. Multicolor flow cytometric analysis of syngeneic lymphoma tumors showed distinct polarization of F4/80+ TAMs into CD206+ M2 and CD80+ M1 phenotypes. Using human MCL cell lines (Mino, Granta, and JVM2), we further showed that MCL cells polarized monocyte-derived macrophages toward an M2-like phenotype, as assessed by CD163+ expression and increased interleukin-10 (IL-10) level; however, levels of the M1 markers CD80 and IL-12 remained unaffected. To show that macrophages contribute to MCL tumorigenesis, we xenografted the human MCL cell line Mino along with CD14+ monocytes and compared tumor growth between these 2 groups. Results showed that xenografted Mino along with CD14+ monocytes significantly increased the tumor growth in vivo compared with MCL cells alone (P < .001), whereas treatment with liposomal clodronate (to deplete the macrophages) reversed the effect of CD14+ monocytes on growth of MCL xenografts (P < .001). Mechanistically, IL-10 secreted by MCL-polarized M2-like macrophages was found to be responsible for increasing MCL growth by activating STAT1 signaling, whereas IL-10 neutralizing antibody or STAT1 inhibition by fludarabine or STAT1 short hairpin RNA significantly abolished MCL growth (P < .01). Collectively, our data show the existence of a tumor microenvironmental network of macrophages and MCL tumor and suggest the importance of macrophages in interventional therapeutic strategies against MCL and other lymphoid malignancies.

Bipotent Progenitors Do Not Require Androgen Receptor for Luminal Specification during Prostate Organogenesis.

Androgen receptor (AR) plays a fundamental role in most aspects of adult prostate homeostasis, and anti-androgen therapy represents the cornerstone of prostate cancer treatment. However, early prostate organogenesis takes place during pre-pubertal stages when androgen levels are low, raising the possibility that AR function is more limited during prostate development. Here, we use inducible AR deletion and lineage tracing in genetically engineered mice to show that basal and luminal epithelial progenitors do not require cell-autonomous AR activity during prostate development. We also demonstrate the existence of a transient bipotent luminal progenitor that can generate luminal and basal progeny, yet is also independent of AR function. Furthermore, molecular analyses of AR-deleted luminal cells isolated from developing prostates indicate their similarity to wild-type cells. Our findings suggest that low androgen levels correlate with luminal plasticity in prostate development and may have implications for understanding how AR inhibition promotes lineage plasticity in prostate cancer.

CRISPR/Cas9-Mediated Point Mutation in Nkx3.1 Prolongs Protein Half-Life and Reverses Effects Nkx3.1 Allelic Loss.

NKX3.1 is the most commonly deleted gene in prostate cancer and is a gatekeeper suppressor. NKX3.1 is haploinsufficient, and pathogenic reduction in protein levels may result from genetic loss, decreased transcription, and increased protein degradation caused by inflammation or PTEN loss. NKX3.1 acts by retarding proliferation, activating antioxidants, and enhancing DNA repair. DYRK1B-mediated phosphorylation at serine 185 of NKX3.1 leads to its polyubiquitination and proteasomal degradation. Because NKX3.1 protein levels are reduced, but never entirely lost, in prostate adenocarcinoma, enhancement of NKX3.1 protein levels represents a potential therapeutic strategy. As a proof of principle, we used CRISPR/Cas9-mediated editing to engineer in vivo a point mutation in murine Nkx3.1 to code for a serine to alanine missense at amino acid 186, the target for Dyrk1b phosphorylation. Nkx3.1S186A/-, Nkx3.1+/- , and Nkx3.1+/+ mice were analyzed over one year to determine the levels of Nkx3.1 expression and effects of the mutant protein on the prostate. Allelic loss of Nkx3.1 caused reduced levels of Nkx3.1 protein, increased proliferation, and prostate hyperplasia and dysplasia, whereas Nkx3.1S186A/- mouse prostates had increased levels of Nkx3.1 protein, reduced prostate size, normal histology, reduced proliferation, and increased DNA end labeling. At 2 months of age, when all mice had normal prostate histology, Nkx3.1+/- mice demonstrated indices of metabolic activation, DNA damage response, and stress response. These data suggest that modulation of Nkx3.1 levels alone can exert long-term control over premalignant changes and susceptibility to DNA damage in the prostate. SIGNIFICANCE: These findings show that prolonging the half-life of Nkx3.1 reduces proliferation, enhances DNA end-labeling, and protects from DNA damage, ultimately blocking the proneoplastic effects of Nkx3.1 allelic loss.

A single-cell atlas of the mouse and human prostate reveals heterogeneity and conservation of epithelial progenitors.

Understanding the cellular constituents of the prostate is essential for identifying the cell of origin for prostate adenocarcinoma. Here, we describe a comprehensive single-cell atlas of the adult mouse prostate epithelium, which displays extensive heterogeneity. We observe distal lobe-specific luminal epithelial populations (LumA, LumD, LumL, and LumV), a proximally enriched luminal population (LumP) that is not lobe-specific, and a periurethral population (PrU) that shares both basal and luminal features. Functional analyses suggest that LumP and PrU cells have multipotent progenitor activity in organoid formation and tissue reconstitution assays. Furthermore, we show that mouse distal and proximal luminal cells are most similar to human acinar and ductal populations, that a PrU-like population is conserved between species, and that the mouse lateral prostate is most similar to the human peripheral zone. Our findings elucidate new prostate epithelial progenitors, and help resolve long-standing questions about anatomical relationships between the mouse and human prostate.

Electrotonic transmission in the retinal vasculature: inhibitory role of the diabetes/VEGF/aPKC pathway.

The deleterious impact of diabetes on the retina is a leading cause of vision loss. Ultimately, the hypoxic retinopathy caused by diabetes results in irreversible damage to vascular, neuronal, and glial cells. Less understood is how retinal physiology is altered early in the course of diabetes. We recently found that the electrotonic architecture of the retinovasculature becomes fundamentally altered soon after the onset of this disorder. Namely, the spread of voltage through the vascular endothelium is markedly inhibited. The goal of this study was to elucidate how diabetes inhibits electrotonic transmission. We hypothesized that vascular endothelial growth factor (VEGF) may play a role since its upregulation in hypoxic retinopathy is associated with sight-impairing complications. In this study, we quantified voltage transmission between pairs of perforated-patch pipettes sealed onto abluminal cells located on retinal microvascular complexes freshly isolated from diabetic and nondiabetic rats. We report that exposure of diabetic retinal microvessels to an anti-VEGF antibody or to a small-molecule inhibitor of atypical PKCs (aPKC) near-fully restored the efficacy of electrotonic transmission. Furthermore, exposure of nondiabetic microvessels to VEGF mimicked, via a mechanism sensitive to the aPKC inhibitor, the diabetes-induced inhibition of transmission. Thus, activation of the diabetes/VEGF/aPKC pathway switches the retinovasculature from a highly interactive operational unit to a functionally balkanized complex. By delimiting the dissemination of voltage-changing vasomotor inputs, this organizational fragmentation is likely to compromise effective regulation of retinal perfusion. Future pharmacological targeting of the diabetes/VEGF/aPKC pathway may serve to impede progression of vascular dysfunction to irreversible diabetic retinopathy.

Purinergic Vasotoxicity: Role of the Pore/Oxidant/KATP Channel/Ca2+ Pathway in P2X7-Induced Cell Death in Retinal Capillaries.

P2X7 receptor/channels in the retinal microvasculature not only regulate vasomotor activity, but can also trigger cells in the capillaries to die. While it is known that this purinergic vasotoxicity is dependent on the transmembrane pores that form during P2X7 activation, events linking pore formation with cell death remain uncertain. To better understand this pathophysiological process, we used YO-PRO-1 uptake, dichlorofluorescein fluorescence, perforated-patch recordings, fura-2 imaging and trypan blue dye exclusion to assess the effects of the P2X7 agonist, benzoylbenzoyl-ATP (BzATP), on pore formation, oxidant production, ion channel activation, [Ca2+]i and cell viability. Experiments demonstrated that exposure of retinal microvessels to BzATP increases capillary cell oxidants via a mechanism dependent on pore formation and the enzyme, NADPH oxidase. Indicative that oxidation plays a key role in purinergic vasotoxicity, an inhibitor of this enzyme completely prevented BzATP-induced death. We further discovered that vasotoxicity was boosted 4-fold by a pathway involving the oxidation-driven activation of hyperpolarizing KATP channels and the resulting increase in calcium influx. Our findings revealed that the previously unappreciated pore/oxidant/KATP channel/Ca2+ pathway accounts for 75% of the capillary cell death triggered by sustained activation of P2X7 receptor/channels. Elucidation of this pathway is of potential therapeutic importance since purinergic vasotoxicity may play a role in sight-threatening disorders such as diabetic retinopathy.

TRIM28 Controls Genomic Imprinting through Distinct Mechanisms during and after Early Genome-wide Reprogramming.

Genomic imprinting depends on the establishment and maintenance of DNA methylation at imprinting control regions. However, the mechanisms by which these heritable marks influence allele-specific expression are not fully understood. By analyzing maternal, zygotic, maternal-zygotic, and conditional Trim28 mutants, we found that the transcription factor TRIM28 controls genomic imprinting through distinct mechanisms at different developmental stages. During early genome-wide reprogramming, both maternal and zygotic TRIM28 are required for the maintenance of methylation at germline imprints. However, in conditional Trim28 mutants, Gtl2-imprinted gene expression was lost despite normal methylation levels at the germline IG-DMR. These results provide evidence that TRIM28 controls imprinting after early embryonic reprogramming through a mechanism other than the maintenance of germline imprints. Additionally, our finding that secondary imprints were hypomethylated in TRIM28 mutants uncovers a requirement of TRIM28 after genome-wide reprogramming for interpreting germline imprints and regulating DNA methylation at imprinted gene promoters.

Stem cells in genetically-engineered mouse models of prostate cancer.

The cancer stem cell model proposes that tumors have a hierarchical organization in which tumorigenic cells give rise to non-tumorigenic cells, with only a subset of stem-like cells able to propagate the tumor. In the case of prostate cancer, recent analyses of genetically engineered mouse (GEM) models have provided evidence supporting the existence of cancer stem cells in vivo. These studies suggest that cancer stem cells capable of tumor propagation exist at various stages of tumor progression from prostatic intraepithelial neoplasia (PIN) to advanced metastatic and castration-resistant disease. However, studies of stem cells in prostate cancer have been limited by available approaches for evaluating their functional properties in cell culture and transplantation assays. Given the role of the tumor microenvironment and the putative cancer stem cell niche, future studies using GEM models to analyze cancer stem cells in their native tissue microenvironment are likely to be highly informative.

Single luminal epithelial progenitors can generate prostate organoids in culture.

The intrinsic ability to exhibit self-organizing morphogenetic properties in ex vivo culture may represent a general property of tissue stem cells. Here we show that single luminal stem/progenitor cells can generate prostate organoids in a three-dimensional culture system in the absence of stroma. Organoids generated from CARNs (castration-resistant Nkx3.1-expressing cells) or normal prostate epithelia exhibit tissue architecture containing luminal and basal cells, undergo long-term expansion in culture and exhibit functional androgen receptor signalling. Lineage-tracing demonstrates that luminal cells are favoured for organoid formation and generate basal cells in culture. Furthermore, tumour organoids can initiate from CARNs after oncogenic transformation and from mouse models of prostate cancer, and can facilitate analyses of drug response. Finally, we provide evidence supporting the feasibility of organoid studies of human prostate tissue. Our studies underscore the progenitor properties of luminal cells, and identify in vitro approaches for studying prostate biology.

Canonical Wnt signaling regulates Nkx3.1 expression and luminal epithelial differentiation during prostate organogenesis.

BACKGROUND: The formation of the prostate gland requires reciprocal interactions between the epithelial and mesenchymal components of the embryonic urogenital sinus. However, the identity of the signaling factors that mediate these interactions is largely unknown. RESULTS: Our studies show that expression of the prostate-specific transcription factor Nkx3.1 is regulated by the canonical Wnt signaling pathway. Using mice carrying a targeted lacZ knock-in allele of Nkx3.1, we find that Nkx3.1 is expressed in all epithelial cells of ductal buds during prostate organogenesis. Addition of Wnt inhibitors to urogenital sinus explant culture greatly reduces prostate budding and inhibits Nkx3.1 expression as well as differentiation of luminal epithelial cells. Analyses of a TCF/Lef:H2B-GFP transgene reporter show that canonical Wnt signaling activity is found in urogenital mesenchyme but not urogenital sinus epithelium before prostate formation, and is later observed in the mesenchyme and epithelium of prostate ductal tips. Furthermore, TCF/Lef:H2B-GFP reporter activity is reduced in epithelial cells of Nkx3.1 null neonatal prostates, suggesting that Nkx3.1 functions to maintain canonical Wnt signaling activity in developing prostate bud tips. CONCLUSIONS: We propose that activated canonical Wnt signals and Nkx3.1 function in a positive feedback loop to regulate prostate bud growth and luminal epithelial differentiation.

The roots of cancer: stem cells and the basis for tumor heterogeneity.

Recent studies of prostate cancer and other tumor types have revealed significant support, as well as unexpected complexities, for the application of concepts from normal stem cell biology to cancer. In particular, the cell of origin and cancer stem cell models have been proposed to explain the heterogeneity of tumors during the initiation, propagation, and evolution of cancer. Thus, a basis of intertumor heterogeneity has emerged from studies investigating whether stem cells and/or non-stem cells can serve as cells of origin for cancer and give rise to tumor subtypes that vary in disease outcome. Furthermore, analyses of putative cancer stem cells have revealed the genetically diverse nature of cancers and expanded our understanding of intratumor heterogeneity and clonal evolution. Overall, the principles that have emerged from these stem cell studies highlight the challenges to be surmounted to develop effective treatment strategies for cancer.

[Relationship between the extent of subarachnoid hemorrhage and intraocular hemorrhages in patients with subarachnoid hemorrhage].

PURPOSE: To examine the relationship between the extent of subarachnoid hemorrhage and intraocular hemorrhages in patients with subarachnoid hemorrhage. SUBJECTS AND METHOD: A total, of 63 patients (25 men and 38 women, mean age 58 years). The subarachnoid hemorrhage quantity was graded according to the Fisher scale and compared with hemorrhages in the ocular fundus. RESULTS: Either vitreous or preretinal hemorrhages in either one or both eyes (vitreous hemorrhages) were present in 16 patients (25%). Retinal hemorrhages in either one or both eyes (retinal hemorrhages) were present in 12 patients (19%). Intraocular hemorrhage was absent in the other 35 patients (56%). The incidence of vitreous hemorrhage tended to be higher than the incidence of retinal hemorrhage or of the absence of hemorrhage as the rate of subarachnoid hemorrhage increased (Kruskal-Wallis, p < 0.05). There was no significant correlation between retinal hemorrhages and the absence of hemorrhage in the Fisher grade IV chi2, p > 0.05). CONCLUSION: The onset of vitreous hemorrhage appears to be related to the extent of subarachnoid hemorrhage but the onset of retinal hemorrhage is not.

Involvement of glial cells in the autoregulation of optic nerve head blood flow in rabbits.

PURPOSE: To investigate the involvement of glial cells in the autoregulation of optic nerve head (ONH) blood flow in response to elevated intraocular pressure (IOP). METHODS: Rabbit eyes were treated with an intravitreal injection of l-2-aminoadipic acid (LAA), a gliotoxic compound. Twenty-four hours after the injection IOP was artificially elevated from a baseline of 20 to 50 or 70 mm Hg and maintained at each IOP level for 30 minutes. ONH blood flow was measured by laser speckle flowgraphy every 10 minutes. Ocular perfusion pressure (OPP) was calculated to investigate the relationship between ONH blood flow and OPP. To evaluate the effects of LAA on the function and morphology of retinal neurons and glial cells, electroretinogram (ERG) was monitored after injections of LAA (2.0 and 6.0 mM) or saline as a control. Histologic and immunohistochemical examinations were then performed. RESULTS: In the LAA-treated eyes, histologic changes selectively occurred in the retinal Müller cells and ONH astrocytes. There was not any significant reduction of amplitude or elongation of implicit time of each parameter in the ERG after LAA injection compared with control. ONH blood flow in LAA-treated eyes was significantly decreased with a reduction of OPP during IOP elevation to 50 and 70 mm Hg, whereas blood flow was maintained in control eyes during IOP elevation to 50 mm Hg. CONCLUSIONS: These results indicate the involvement of glial ells in the autoregulation of ONH blood flow during IOP elevation.