The total number of glomeruli is a fundamental parameter of kidney function but very difficult to determine using standard methodology. Here, we counted all individual glomeruli in murine kidneys and sized the capillary tufts by combining in vivo fluorescence labeling of endothelial cells, a novel tissue-clearing technique, lightsheet microscopy, and automated registration by image analysis. Total hands-on time per organ was <1 hour, and automated counting/sizing was finished in <3 hours. We also investigated the novel use of ethyl-3-phenylprop-2-enoate (ethyl cinnamate) as a nontoxic solvent-based clearing reagent that can be handled without specific safety measures. Ethyl cinnamate rapidly cleared all tested organs, including calcified bone, but the fluorescence of proteins and immunohistochemical labels was maintained over weeks. Using ethyl cinnamate-cleared kidneys, we also quantified the average creatinine clearance rate per glomerulus. This parameter decreased in the first week of experimental nephrotoxic nephritis, whereas reduction in glomerular numbers occurred much later. Our approach delivers fundamental parameters of renal function, and because of its ease of use and speed, it is suitable for high-throughput analysis and could greatly facilitate studies of the effect of kidney diseases on whole-organ physiology.

Neuronal injury from ischemic stroke is aggravated by invading peripheral immune cells. Early infiltrates of neutrophil granulocytes and T-cells influence the outcome of stroke. So far, however, neither the timing nor the cellular dynamics of neutrophil entry, its consequences for the invaded brain area, or the relative importance of T-cells has been extensively studied in an intravital setting. Here, we have used intravital two-photon microscopy to document neutrophils and brain-resident microglia in mice after induction of experimental stroke. We demonstrated that neutrophils immediately rolled, firmly adhered, and transmigrated at sites of endothelial activation in stroke-affected brain areas. The ensuing neutrophil invasion was associated with local blood-brain barrier breakdown and infarct formation. Brain-resident microglia recognized both endothelial damage and neutrophil invasion. In a cooperative manner, they formed cytoplasmic processes to physically shield activated endothelia and trap infiltrating neutrophils. Interestingly, the systemic blockade of very-late-antigen-4 immediately and very effectively inhibited the endothelial interaction and brain entry of neutrophils. This treatment thereby strongly reduced the ischemic tissue injury and effectively protected the mice from stroke-associated behavioral impairment. Behavioral preservation was also equally well achieved with the antibody-mediated depletion of myeloid cells or specifically neutrophils. In contrast, T-cell depletion more effectively reduced the infarct volume without improving the behavioral performance. Thus, neutrophil invasion of the ischemic brain is rapid, massive, and a key mediator of functional impairment, while peripheral T-cells promote brain damage. Acutely depleting T-cells and inhibiting brain infiltration of neutrophils might, therefore, be a powerful early stroke treatment.

GM1-ganglioside (GM1) is a major sialoglycolipid of neuronal membranes that, among other functions, modulates calcium homeostasis. Excessive accumulation of GM1 due to deficiency of lysosomal beta-galactosidase (beta-gal) characterizes the neurodegenerative disease GM1-gangliosidosis, but whether the accumulation of GM1 is directly responsible for CNS pathogenesis was unknown. Here we demonstrate that activation of an unfolded protein response (UPR) associated with the upregulation of BiP and CHOP and the activation of JNK2 and caspase-12 leads to neuronal apoptosis in the mouse model of GM1-gangliosidosis. GM1 loading of wild-type neurospheres recapitulated the phenotype of beta-gal-/- cells and activated this pathway by depleting ER calcium stores, which ultimately culminated in apoptosis. Activation of UPR pathways did not occur in mice double deficient for beta-gal and ganglioside synthase, beta-gal-/-/GalNAcT-/-, which do not accumulate GM1. These findings suggest that the UPR can be induced by accumulation of the sialoglycolipid GM1 and this causes a novel mechanism of neuronal apoptosis.

Neutrophil granulocyte biology is a central issue of immunological research, but the lack of animal models that allow for neutrophil-selective genetic manipulation has delayed progress. By modulating the neutrophil-specific locus Ly6G with a knock-in allele expressing Cre recombinase and the fluorescent protein tdTomato, we generated a mouse model termed Catchup that exhibits strong neutrophil specificity. Transgene activity was found only in very few eosinophils and basophils and was undetectable in bone marrow precursors, including granulomonocytic progenitors (GMPs). Cre-mediated reporter-gene activation allowed for intravital two-photon microscopy of neutrophils without adoptive transfer. Homozygous animals were Ly6G deficient but showed normal leukocyte cellularity in all measured organs. Ly6G-deficient neutrophils were functionally normal in vitro and in multiple models of sterile or infectious inflammation in vivo. However, Cre-mediated deletion of FcγRIV in neutrophils reduced the cells' recruitment to immune-complex-mediated peritonitis, suggesting a cell-intrinsic role for activating Fc receptors in neutrophil trafficking.