Versatile cell ablation tools and their applications to study loss of cell functions.

Targeted cell ablation is a powerful approach for studying the role of specific cell populations in a variety of organotypic functions, including cell differentiation, and organ generation and regeneration. Emerging tools for permanently or conditionally ablating targeted cell populations and transiently inhibiting neuronal activities exhibit a diversity of application and utility. Each tool has distinct features, and none can be universally applied to study different cell types in various tissue compartments. Although these tools have been developed for over 30 years, they require additional improvement. Currently, there is no consensus on how to select the tools to answer the specific scientific questions of interest. Selecting the appropriate cell ablation technique to study the function of a targeted cell population is less straightforward than selecting the method to study a gene's functions. In this review, we discuss the features of the various tools for targeted cell ablation and provide recommendations for optimal application of specific approaches.

Salt-sensitive increase in macrophages in the kidneys of Dahl SS rats.

Studies of Dahl salt-sensitive (SS) rats have shown that renal CD3+ T cells and ED-1+ macrophages are involved in the development of salt-sensitive hypertension and renal damage. The present study demonstrated that the increase in renal immune cells, which accompanies renal hypertrophy and albuminuria in high-salt diet-fed Dahl SS rats, is absent in Sprague-Dawley and SSBN13 rats that are protected from the SS disease phenotype. Flow cytometric analysis demonstrated that >70% of the immune cells in the SS kidney are M1 macrophages. PCR profiling of renal myeloid cells showed a salt-induced upregulation in 9 of 84 genes related to Toll-like receptor signaling, with notable upregulation of the Toll-like receptor 4/CD14/MD2 complex. Because of the prominent increase in macrophages in the SS kidney, we used liposome-encapsulated clodronate (Clod) to deplete macrophages and assess their contribution to salt-sensitive hypertension and renal damage. Dahl SS animals were administered either Clod-containing liposomes (Clod-Lipo), Clod, or PBS-containing liposomes as a vehicle control. Clod-Lipo treatment depleted circulating and splenic macrophages by ∼50%; however, contrary to our hypothesis, Clod-Lipo-treated animals developed an exacerbated salt-sensitive response with respect to blood pressure and albuminuria, which was accompanied by increased renal T and B cells. Interestingly, those treated with Clod also demonstrated an exacerbated phenotype, but it was less severe than Clod-Lipo-treated animals and independent of changes to the number of renal immune cells. Here, we have shown that renal macrophages in Dahl SS animals sustain a M1 proinflammatory phenotype in response to increased dietary salt and highlighted potential adverse effects of Clod-Lipo macrophage depletion.

Macrophage Populations and Expression of Regulatory Inflammatory Factors in Hepatic Macrophage-depleted Rat Livers under Lipopolysaccharide (LPS) Treatment.

To investigate the significance of the appearance of hepatic macrophages and expression of inflammatory factors in normal and macrophage-depleted livers, hepatic macrophages were depleted with liposome (Lipo)-encapsulated clodronate (CLD; 50 mg/kg, i.v.) followed by lipopolysaccharide (LPS) administration (0.1 mg/kg, i.p.) in F344 rats (CLD + LPS). Vehicle control rats (Lipo + LPS) received empty-Lipo before LPS. The low dose of LPS did not result in microscopic changes in the liver in either treatment group but did modulate M1 and M2 macrophage activity in Lipo + LPS rats without altering repopulating hepatic macrophages in CLD + LPS rats. LPS treatment in Lipo + LPS rats dramatically increased the M1 (IL-1ß, IL-6, TNF-α, and MCP-1) but not M2 macrophage-related factors (IL-4 and CSF-1) compared to CLD + LPS rats. In the CLD + LPS rats, the M2 macrophage-related factors IL-4 and CSF-1 were elevated. In conclusion, low-dose LPS activated hepatic macrophages in rat livers without causing liver injury or stimulating repopulating hepatic macrophages. These data suggest that LPS may alter the liver microenvironment by modulating M1 or M2 macrophage-related inflammatory mediators and macrophage-based hepatotoxicity.

Microglial Cells Prevent Hemorrhage in Neonatal Focal Arterial Stroke.

Perinatal stroke leads to significant morbidity and long-term neurological and cognitive deficits. The pathophysiological mechanisms of brain damage depend on brain maturation at the time of stroke. To understand whether microglial cells limit injury after neonatal stroke by preserving neurovascular integrity, we subjected postnatal day 7 (P7) rats depleted of microglial cells, rats with inhibited microglial TGFbr2/ALK5 signaling, and corresponding controls, to transient middle cerebral artery occlusion (tMCAO). Microglial depletion by intracerebral injection of liposome-encapsulated clodronate at P5 significantly reduced vessel coverage and triggered hemorrhages in injured regions 24 h after tMCAO. Lack of microglia did not alter expression or intracellular redistribution of several tight junction proteins, did not affect degradation of collagen IV induced by the tMCAO, but altered cell types producing TGFß1 and the phosphorylation and intracellular distribution of SMAD2/3. Selective inhibition of TGFbr2/ALK5 signaling in microglia via intracerebral liposome-encapsulated SB-431542 delivery triggered hemorrhages after tMCAO, demonstrating that TGFß1/TGFbr2/ALK5 signaling in microglia protects from hemorrhages. Consistent with observations in neonatal rats, depletion of microglia before tMCAO in P9 Cx3cr1(GFP/+)/Ccr2(RFP/+) mice exacerbated injury and induced hemorrhages at 24 h. The effects were independent of infiltration of Ccr2(RFP/+) monocytes into injured regions. Cumulatively, in two species, we show that microglial cells protect neonatal brain from hemorrhage after acute ischemic stroke.

Macrophage depletion impairs skeletal muscle regeneration: The roles of regulatory factors for muscle regeneration.

Though macrophages are essential for skeletal muscle regeneration, which is a complex process, the roles and mechanisms of the macrophages in the process of muscle regeneration are still not fully understood. The objective of this study is to explore the roles of macrophages and the mechanisms involved in the regeneration of injured skeletal muscle. One hundred and twelve C57BL/6 mice were randomly divided into muscle contusion and macrophages depleted groups. Their gastrocnemius muscles were harvested at the time points of 12 h, 1, 3, 5, 7, 14 d post-injury. The changes in skeletal muscle morphology were assessed by hematoxylin and eosin (HE) stain. The gene expression was analyzed by real-time polymerase chain reaction. The data showed that CL-liposomes treatment did affect the expression of myogenic regulatory factors (MyoD, myogenin) after injury. In addition, CL-liposomes treatment decreased the expression of regulatory factors of muscle regeneration (HGF, uPA, COX-2, IGF-1, MGF, FGF6) and increased the expression of inflammatory cytokines (TGF-ß1, TNF-α, IL-1ß, RANTES) in the late stage of regeneration. Moreover, there were significant correlations between macrophages and some regulatory factors (such as HGF, uPA) for muscle regeneration. These results suggested that macrophages depletion impairs skeletal muscle regeneration and that the regulatory factors for muscle regeneration may play important roles in this process.

Cutting edge: nitrogen bisphosphonate-induced inflammation is dependent upon mast cells and IL-1.

Nitrogen-containing bisphosphonates (NBPs) are taken by millions for bone disorders but may cause serious inflammatory reactions. In this study, we used a murine peritonitis model to characterize the inflammatory mechanisms of these agents. At dosages comparable to those used in humans, injection of NBPs into the peritoneum caused recruitment of neutrophils, followed by an influx of monocytes. These cellular changes corresponded to an initial increase in IL-1α, which preceded a rise in multiple other proinflammatory cytokines. IL-1R, IL-1α, and IL-1ß were required for neutrophil recruitment, whereas other MyD88-dependent signaling pathways were needed for the monocyte influx. Mice deficient in mast cells, but not mice lacking lymphocytes, were resistant to NBP-induced inflammation, and reconstitution of these mice with mast cells restored sensitivity to NBPs. These results document the critical role of mast cells and IL-1 in NBP-mediated inflammatory reactions.

Neuroprotective function for ramified microglia in hippocampal excitotoxicity.

BACKGROUND: Most of the known functions of microglia, including neurotoxic and neuroprotective properties, are attributed to morphologically-activated microglia. Resting, ramified microglia are suggested to primarily monitor their environment including synapses. Here, we show an active protective role of ramified microglia in excitotoxicity-induced neurodegeneration. METHODS: Mouse organotypic hippocampal slice cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic neuronal cell death. This procedure was performed in slices containing resting microglia or slices that were chemically or genetically depleted of their endogenous microglia. RESULTS: Treatment of mouse organotypic hippocampal slice cultures with 10-50 µM N-methyl-D-aspartic acid (NMDA) induced region-specific excitotoxic neuronal cell death with CA1 neurons being most vulnerable, whereas CA3 and DG neurons were affected less. Ablation of ramified microglia severely enhanced NMDA-induced neuronal cell death in the CA3 and DG region rendering them almost as sensitive as CA1 neurons. Replenishment of microglia-free slices with microglia restored the original resistance of CA3 and DG neurons towards NMDA. CONCLUSIONS: Our data strongly suggest that ramified microglia not only screen their microenvironment but additionally protect hippocampal neurons under pathological conditions. Morphological activation of ramified microglia is thus not required to influence neuronal survival.

Monocyte/macrophages promote vasculogenesis in choroidal neovascularization in mice by stimulating SDF-1 expression in RPE cells.

BACKGROUND: Monocyte-macrophages play important roles in choroidal neovascularization (CNV); however, the mechanism is unclear. This study investigated the effects of monocyte depletion on laser-induced CNV in mice, especially the involvement of bone marrow-derived cells (BMCs) and underlying molecular mechanisms. METHODS: Clodronate-liposomes (lip) were used to deplete monocytes and their effect on retinal pigmental epithelium (RPE) cells, endothelial cells, and BMCs was analyzed. Green fluorescent protein (GFP)-chimeric mice were developed by transplanting bone marrow cells from GFP transgenic mice to C57BL/6 J mice. CNV was induced by laser photocoagulation. Chimeric mice were intravenously treated with clodronate-lip, PBS-lip or PBS, 1 day before and after lasering. Histopathological and choroidal flatmount analysis were performed to measure CNV severity and BMCs recruitment. BMCs expression of endothelial cell marker CD31 and vascular smooth muscle cell marker α-SMA in CNV were detected by immunofluorescence. Expression of stromal cell-derived factor-1 (SDF-1) protein in vivo was detected by immunofluorescence as well as ELISA assay. SDF-1 was also examined by RT-PCR and ELISA in a human monocytes-RPE cells co-culturing system. RESULTS: No valid evidence for the toxicity of clodronate-lip was found. Depletion led to significant inhibition of CNV and BMCs recruitment into laser spots on days 3 and 14, reduced BMC expression of CD31 and α-SMA on day 14, and decreased expression of SDF-1 in vivo on day 3. SDF-1 was mostly within and around the RPE cells in the laser lesion. SDF-1 was dramatically up-regulated in RPE cells after co-culturing with monocytes. CONCLUSIONS: Monocytes may promote experimental CNV, especially BMC contribution in mice, by promoting SDF-1 production in RPE cells.

Bisphosphonates: restrictions for vasculogenesis and angiogenesis: inhibition of cell function of endothelial progenitor cells and mature endothelial cells in vitro.

Bisphosphonate-associated osteonecrosis of the jaws (BP-ONJ) is one of the main side effects in patients treated with bisphosphonates for metastasis to the bone or osteoporosis. BP-ONJ usually occurs in patients treated with highly potent nitrogen-containing bisphosphonates. The exact mechanism of action and etiopathology is still unknown. In addition to inhibition of bone remodelling, an anti-angiogenetic effect has become the focus of research. The aim of these study was to investigate the effect of different bisphosphonates on human umbilicord vein endothelial cells (HUVEC) and endothelial progenitor cells (EPC), which play an important role in angiogenesis. Using varying concentrations, the impact of one non-nitrogen-containing bisphosphonate (clodronate) and three nitrogen-containing bisphosphonates (ibandronate, pamidronate and zoledronate) on HUVEC and EPC was analysed. The biologic behaviour of HUVEC after incubation with different bisphosphonates was measured in a Boyden migration assay as well as in a 3D angiogenesis assay. The number of apoptotic cells was measured by Tunnel assay. To underline the importance of neoangiogenesis in the context of BP-ONJ, we measured the EPC number after incubation with different bisphosphonates in vitro. HUVEC and EPC were significantly influenced by bisphosphonates at different concentrations compared with the non-treated control groups. The nitrogen-containing bisphosphonates pamidronate and zoledronate had the greatest impact on the cells, whereas clodronate followed by ibandronate was less distinct on cell function. These results underline the hypothesis that inhibited angiogenesis induced by bisphosphonates might be of relevance in the development and maintenance of BP-ONJ. The increased impact by highly potent bisphosphonates on HUVEC and EPC may explain the high prevalence of BP-ONJ in patients undergoing this treatment.

Cytotoxic effect of clodronate and zoledronate on the chondrosarcoma cell lines HTB-94 and CAL-78.

The wide surgical tumour resection is the only effective treatment in chondrosarcoma. However, a major problem remains the high rate of local recurrences and metastases due to the lack of adjuvant therapies. In this study the cytotoxic effect of the bisphosphonate clodronate (0.1-1000 µM) and zoledronate (0.1-1000 µM) in different concentrations on two chondrosarcoma cell lines (HTB-94 and CAL-78) has been investigated. After an incubation period of 48, 72 and 96 hours the chondrosarcoma cell viability was measured as the MTT-proliferation rate. In concentrations of >1 µm zoledronate the cell activity was reduced by up to 95% for the CAL-78 cells. Further, zoledronate has been more effective in lower concentrations than clodronate in the reduction of cell viability for both cell lines. However, clodronate showed significant cytotoxic effects in high concentrations and after longer incubation periods. Further research is necessary, but in the light of these results bisphosphonates may also play a role in the treatment of chondrosarcomas.

Resident macrophages influence stem cell activity in the mammary gland.

INTRODUCTION: Macrophages in the mammary gland are essential for morphogenesis of the ductal epithelial tree and have been implicated in promoting breast tumor metastasis. Although it is well established that macrophages influence normal mammopoiesis, the mammary cell types that these accessory cells influence have not been determined. Here we have explored a role for macrophages in regulating mammary stem cell (MaSC) activity, by assessing the ability of MaSCs to reconstitute a mammary gland in a macrophage-depleted fat pad. METHODS: Two different in vivo models were used to deplete macrophages from the mouse mammary fat pad, allowing us to examine the effect of macrophage deficiency on the mammary repopulating activity of MaSCs. Both the Csf1op/op mice and clodronate liposome-mediated ablation models entailed transplantation studies using the MaSC-enriched population. RESULTS: We show that mammary repopulating ability is severely compromised when the wild-type MaSC-enriched subpopulation is transplanted into Csf1op/op fat pads. In reciprocal experiments, the MaSC-enriched subpopulation from Csf1op/op glands had reduced regenerative capacity in a wild-type environment. Utilizing an alternative strategy for selective depletion of macrophages from the mammary gland, we demonstrate that co-implantation of the MaSC-enriched subpopulation with clodronate-liposomes leads to a marked decrease in repopulating frequency and outgrowth potential. CONCLUSIONS: Our data reveal a key role for mammary gland macrophages in supporting stem/progenitor cell function and suggest that MaSCs require macrophage-derived factors to be fully functional. Macrophages may therefore constitute part of the mammary stem cell niche.

Effects of Kupffer cell-depletion on Concanavalin A-induced hepatitis.

TNF-alpha, IFN-gamma, IL-4, and MIP-2 are known to be involved in Con A-induced hepatitis. Although Kupffer cells are reportedly involved in TNF-alpha production, it is largely unknown whether or not Kupffer cells also play a role in the production of other cytokines, such as IFN-gamma, IL-4, and MIP-2. In this study we examined the liver injury and the levels of plasma cytokines, including above four cytokines, KC, and IL-10 in Kupffer cell-depleted mice obtained through administration of liposome-encapsulated dichloromethylene bisphosphonate. The liver injury was significantly suppressed in Kupffer cell-depleted mice, as assessed as to the plasma ALT level and histochemistry. The cytokine levels were also significantly suppressed in such mice except for those of IFN-gamma, which was slightly suppressed at 12h, and IL-10, which was not significantly suppressed at any time. Apoptosis was also significantly suppressed in such mice, as found immunohistochemically with anti-ssDNA Ab. Taken together, these results suggest that Kupffer cells are involved in the production of MIP-2, KC, IL-4, and TNF-alpha in Con A-induced hepatitis, thereby contributing to the liver injury either directly or indirectly.

Preclinical evidence for nitrogen-containing bisphosphonate inhibition of farnesyl diphosphate (FPP) synthase in the kidney: implications for renal safety.

Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and play an important role in the treatment of osteoporosis, metastatic bone disease, and Paget disease. However, nephrotoxicity has been reported with some bisphosphonates. Nitrogen-containing bisphosphonates directly inhibit farnesyl diphosphate (FPP) synthase activity (mevalonate pathway) and reduce protein prenylation leading to osteoclast cell death. The aim here was to elucidate if this inhibition also occurs in kidney cells and may directly account for nephrotoxicity. In an exploratory study in rats receiving zoledronate or ibandronate an approximate 2-fold increase in FPP synthase mRNA levels was observed in the kidney. The involvement of the mevalonate pathway was confirmed in subsequent in vitro studies with zoledronate, ibandronate, and pamidronate, using the non-nitrogen containing bisphosphonate clodronate as a comparator. In vitro changes in FPP synthase mRNA expression, enzyme activity, and levels of prenylated proteins were assessed. Using two cell lines (a rat normal kidney cell line, NRK-52E, and a human kidney proximal tubule cell line, HK-2), ibandronate and zoledronate were identified as most cytotoxic (EC50: 23/>1000 microM and 16/82 microM, respectively) and as the most potent inhibitors of FPP synthase (IC50; 1.6/7.4 microM and 0.5/0.7 microM, respectively). In both cell lines, inhibition of FPP synthase activity occurred prior to a decrease in levels of prenylated proteins followed by cytotoxicity. This further supports that the mechanism responsible for osteoclast inhibition (therapeutic effect) might also underlie the mechanism of nephrotoxicity.

Inhibition by diphosphonates of bone resorption induced by the Walker tumor of the rat.

An animal model is described to test the effect of diphosphonates, which are powerful antiosteolytic agents, against bone tumors. This model consists of injecting Walker tumor cells into one iliac artery of a series of rats while the contralateral artery is clamped during the injection, and waiting 7 days to obtain a significant destruction of the femur and tibia of the rats. In most of the animals, after this delay, extensive lesions are observed macroscopically by X-ray and histologically. The parenteral administration of three diphosphonates, dichloromethylene diphosphonate, ethanehydroxydiphosphonate , and aminopropanediphosphonate , at 16 and 160 mumol/kg/day, protects the bones by decreasing the extent of osteolysis. This protective effect is seen both in the tumor-injected leg and in the contralateral leg and is significant when compared to nontreated animals. The most active of the drugs was dichloromethylene-diphosphonate; ethanehydroxydiphosphonate and aminopropanediphosphonate were less active, especially when given at the higher dosage. All diphosphonates produce a marked decrease of the number of osteoclasts; ethanehydroxydiphosphonate at the higher dosage, induced a large increase of nonmineralized bone. These results are discussed in light of recent clinical work, showing that this animal model is a useful tool to test the effect of new drugs against osteolysis of cancer.

Immunophenotypical characterization and influence on liver homeostasis of depleting and repopulating hepatic macrophages in rats injected with clodronate.

Hepatic macrophages (including Kupffer cells) play a crucial role in the homeostasis and act as mediators of inflammatory response in the liver. Hepatic macrophages were depleted in male F344 rats by a single intravenous injection of liposomal clodronate (CLD; 50mg/kg body weight), and immunophenotypical characteristics of depleting and repopulating macrophages were analyzed by different antibodies specific for macrophages. CD163(+) Kupffer cells were almost completely depleted on post-injection (PI) days 1-12. Macrophages reacting to CD68, Iba-1, and Gal-3 were drastically reduced in number on PI day 1 and then recovered gradually until PI day 12. MHC class II(+) and CD204(+) macrophages were moderately decreased during the observation period. Although hepatic macrophages detectable by different antibodies were reduced in varying degrees, Kupffer cells were the most susceptible to CLD. Liver situation influenced by depleted hepatic macrophages was also investigated. No marked histological changes were seen in the liver, but the proliferating activity of hepatocytes was significantly increased, supported by changes of gene profiles relating to cell proliferation on microarray analysis on PI day 1; the values of AST and ALT were significantly elevated; macrophage induction/activation factors (such as MCP-1, CSF-1, IL-6 and IL-4) were increased exclusively on PI day 1, whereas anti-inflammatory factors such as IL-10 and TGF-ß1 remained significantly decreased after macrophage depletion. The present study confirmed importance of hepatic macrophages in liver homeostasis. The condition of hepatic macrophages should be taken into consideration when chemicals capable of inhibiting macrophage functions are evaluated.

Impaired splenic erythropoiesis in phlebotomized mice injected with CL2MDP-liposome: an experimental model for studying the role of stromal macrophages in erythropoiesis.

Erythropoiesis occurs in the presence of erythropoietin (EPO) without macrophages in vitro. In hematopoietic tissues, however, erythroid cells associate closely with stromal macrophages, forming erythroblastic islands via interactions with adhesion molecules. To elucidate the role of macrophages in erythropoiesis, we selectively abrogated stromal macrophages of splenic red pulp of phlebotomized mice by injection with dichloromethylene diphosphonate encapsulated in multilamellar liposomes (CL2MDP-liposome). In the spleen, no erythropoietic activity occurred until 5 days after the treatment. Colony assay revealed that the erythropoiesis was suppressed at the level of CFU-E. The splenic erythropoietic activity gradually developed from day 6 after the treatment, when F4/80+ macrophages began to appear in the red pulp. EPO mRNA was expressed in kidney but not in liver or spleen of phlebotomized mice injected with CL2MDP-liposome, and the serum EPO concentration in these mice was higher than that in phlebotomized mice. These findings suggest that abrogation of stromal macrophages by injection with CL2MDP-liposome impairs the splenic microenvironment for erythropoiesis induced by hypoxic stress, and this may be an excellent experimental model for further characterization of the in vivo role of splenic macrophages in erythropoiesis.

Cytotoxicity of dichloromethane diphosphonate and of 1-hydroxyethane-1,1-diphosphonate in the amoebae of the slime mould Dictyostelium discoideum. A 31P NMR study.

Two pyrophosphate analogues, dichloromethane diphosphonate (Cl2MDP), and 1-hydroxyethane-1,1-diphosphonate (EHDP), at concentrations of 0.5-1 mM, efficiently inhibited the growth of amoebae of the slime mould Dictyostelium discoideum. Cell viability decreased markedly upon incubation with the diphosphonates. The mechanism of toxicity was investigated by in vivo 31P NMR spectroscopy and the formation of analogues of ATP [adenosine 5'-(beta, gamma-dichloromethane triphosphate) and adenosine 5'-(beta, gamma-1-hydroxyethane triphosphate)] was demonstrated. These two compounds were identified from their 31P NMR spectra in perchloric acid extracts prepared from amoebae poisoned with Cl2MDP or EHDP and may have been synthesized by reversible pyrophosphate exchange catalysed by cytosolic aminoacyl-tRNA synthetases.

Response to Leydig cell apoptosis in the absence of testicular macrophages.

Removal of apoptotic cells from the tissues appears to be a major function of resident tissue macrophages. In order to investigate further the role of testicular macrophages after massive Leydig cell death, adult rats were injected intra-testicularly with liposome-entrapped dichloromethylene diphosphonate (Cl2MDP-lp, right testis) to deplete testicular macrophages, and with NaCl (left testis) as control. Ten days later, the animals were injected intraperitoneally with ethylene dimethane sulphonate (EDS) to induce Leydig cell apoptosis. In macrophage-containing testes there was a 2-fold increase in the number of macrophages on days 1-3 after EDS treatment and Leydig cells were completely eliminated from the interstitium by the second day after treatment. The main differences in the response to Leydig cell death in macrophage-depleted testes were: (1) an early rise in the concentration of small mononuclear, lymphocyte-like cells, (2) a greater influx of circulating monocytes, (3) the existence of variable inflammatory infiltrates on days 3-4, and (4) the disappearance of infiltrating monocytes by day 10. These results suggest that resident macrophages prevent the inflammatory reaction elicited by massive Leydig cell death.

Effects of macrophage depletion at different times after treatment with ethylene dimethane sulfonate (EDS) on the regeneration of Leydig cells in the adult rat.

Testicular macrophages were selectively depleted in the right testes of adult rats by an intratesticular injection of dichloromethylene diphosphonate-containing liposomes (Cl2MDP-lp), whereas the left testes were injected with 0.9% NaCl and served as control. Before or after Leydig cell destruction with ethylene dimethane sulfonate (EDS), treatment with Cl2MDP-lp/NaCl was given at different times to study the requirements of macrophages in the different stages of Leydig cell regeneration. On day 30 after EDS treatment, new Leydig cells were abundant in the left, macrophage-containing testes. However, in the right, macrophage-depleted testes, the number of Leydig cells was related to the time elapsed between EDS treatment and macrophage depletion. When macrophages were depleted on day 10 before or on days 4 or 10 after EDS treatment, new Leydig cells were nearly absent at 30 days. However, when macrophages were depleted on days 16 or 22 after EDS treatment, Leydig cells were found at 30 days, but their numbers were equivalent to the number of Leydig cells that were already present in EDS-treated animals at the time the macrophages were depleted. These results indicate that macrophages are needed for the differentiation of Leydig cells from mesenchymal precursors, as well as for the proliferative activity of the newly formed Leydig cells, possibly through the secretion of essential growth factors.

The reactivity, distribution and abundance of Non-astrocytic Inner Retinal Glial (NIRG) cells are regulated by microglia, acute damage, and IGF1.

Recent studies have described a novel type of glial cell that is scattered across the inner layers of the avian retina and possibly the retinas of primates. These cells have been termed Non-astrocytic Inner Retinal Glial (NIRG) cells. These cells are stimulated by insulin-like growth factor 1 (IGF1) to proliferate, migrate distally into the retina, and become reactive. These changes in glial activity correlate with increased susceptibility of retinal neurons and Müller glia to excitotoxic damage. The purpose of this study was to further study the NIRG cells in retinas treated with IGF1 or acute damage. In response to IGF1, the reactivity, proliferation and migration of NIRG cells persists through 3 days after treatment. At 7 days after treatment, the numbers and distribution of NIRG cells returns to normal, suggesting that homeostatic mechanisms are in place within the retina to maintain the numbers and distribution of these glial cells. By comparison, IGF1-induced microglial reactivity persists for at least 7 days after treatment. In damaged retinas, we find a transient accumulation of NIRG cells, which parallels the accumulation of reactive microglia, suggesting that the reactivity of NIRG cells and microglia are linked. When the microglia are selectively ablated by the combination of interleukin 6 and clodronate-liposomes, the NIRG cells down-regulate transitin and perish within the following week, suggesting that the survival and phenotype of NIRG cells are somehow linked to the microglia. We conclude that the abundance, reactivity and retinal distribution of NIRG cells can be dynamic, are regulated by homoestatic mechanisms and are tethered to the microglia.