Septic encephalopathy with confusion and agitation occurs early during sepsis and contributes to the severity of the disease. A decrease in the sphingosine-1-phosphate (S1P) blood levels has been shown in patients and in animal models of sepsis. The lipid mediator S1P is known to be involved in endothelial barrier function in a context-dependent manner. We utilized lipopolysaccharide (LPS)-injected mice as a model for septic encephalopathy and first performed tracer permeability assays to assess the blood-brain barrier (BBB) breakdown in vivo. At time points corresponding to the BBB breakdown post LPS injection, we aimed to characterize the regulation of the sphingolipid signaling pathway at the BBB during sepsis. We measured sphingolipid concentrations in blood, in mouse brain microvessels (MBMVs), and brain tissue. We also analyzed the expression of S1P receptors, transporters, and metabolizing enzymes in MBMVs and brain tissue. Primary mouse brain microvascular endothelial cells (MBMECs) were isolated to evaluate the effects of LPS on transendothelial electrical resistance (TEER) as a measure of permeability in vitro. We observed a relevant decrease in S1P levels after LPS injection in all three compartments (blood, MBMVs, brain tissue) that was accompanied by an increased expression of the S1P receptor type 1 and of sphingosine kinase 1 on one hand and of the S1P degrading enzymes lipid phosphate phosphatase 1 (LPP1) and S1P phosphatase 1 on the other hand, as well as a down-regulation of sphingosine kinase 2. Application of LPS to a monolayer of primary MBMECs did not alter TEER, but serum from LPS-treated mice lead to a breakdown of the barrier compared to serum from vehicle-treated mice. We observed profound alterations of the sphingolipid metabolism at the BBB after LPS injection that point toward a therapeutic potential of drugs interfering with this pathway as novel approach for the detrimental overwhelming immune response in sepsis. Read the Editorial Highlight for this article on page 115. Cover Image for this Issue: doi. 10.1111/jnc.14161.
Vascular endothelial growth factor A (VEGFA) isoform treatment has been demonstrated to alter spermatogonial stem cell homeostasis. Therefore, we generated pDmrt1-Cre;Vegfa(-/-) (knockout, KO) mice by crossing pDmrt1-Cre mice to floxed Vegfa mice to test whether loss of all VEGFA isoforms in Sertoli and germ cells would impair spermatogenesis. When first mated, KO males took 14 days longer to get control females pregnant (P < .02) and tended to take longer for all subsequent parturition intervals (9 days; P < .07). Heterozygous males sired fewer pups per litter (P < .03) and after the first litter took 10 days longer (P < .05) to impregnate females, suggesting a more progressive loss of fertility. Reproductive organs were collected from 6-month-old male mice. There were fewer sperm per tubule in the corpus epididymides (P < .001) and fewer ZBTB16-stained undifferentiated spermatogonia (P < .003) in the testes of KO males. Testicular mRNA abundance for Bcl2 (P < .02), Bcl2:Bax (P < .02), Neurog3 (P < .007), and Ret was greater (P = .0005), tended to be greater for Sin3a and tended to be reduced for total Foxo1 (P < .07) in KO males. Immunofluorescence for CD31 and VE-Cadherin showed no differences in testis vasculature; however, CD31-positive staining was evident in undifferentiated spermatogonia only in KO testes. Therefore, loss of VEGFA isoforms in Sertoli and germ cells alters genes necessary for long-term maintenance of undifferentiated spermatogonia, ultimately reducing sperm numbers and resulting in subfertility.