R system and the reaction mixtures were incubated at 95C for 10 min, followed by 40 cycles of 95C for 15 s and 60C for 1 min. The threshold cycle values were calculated with QuanStudio Software version 1.2.2. The housekeeping gene Gapdh, and Actb were included in the analysis as controls, and water was included as a negative control. Fold change in the gene was calculated by the equation 2-Ct, the expression was normalized by the housekeeping gene Gapdh, using DataAssist software version 1.2.2. Immunohistochemistry P22 Gjb1-/Y//Gjc2-/- mice PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850275 and their Gjb1+/Y//Gjc2+/+ littermates were perfused with PBS followed by 4% paraformaldehyde in PBS, the cerebra and cerebella were dissected and fixed for another hour, infiltrated overnight in 10% sucrose in PBS at 4C, then embedded in OCT. Cryostat sections were thaw-mounted on Super Frost Plus glass slides and stored at -20C. A spleen was dissected and processed in a similar way and used as a control through the immunostaining procedures. Tissue sections were permeabilized by immersion in -20C acetone for 10 min, incubated for 1 h in blocking solution, incubated overnight at 4C with one of the following antibodies: a rat monoclonal antibody Author Manuscript Author Manuscript Author Manuscript Author Manuscript Neurobiol Dis. Author manuscript; available in PMC 2016 October 01. Wasseff and Scherer Page 4 against Ly6c, a rabbit antisera against CD3, CD72, glial fibrillary acid protein, or Iba1, washed several times in PBS, incubated with rhodamine-conjugated donkey anti-mouse, anti-rat, or anti-rabbit antisera, washed in PBS, mounted with Vectashield with DAPI, and examined by epifluorescence with appropriate optical filters. The mRNA level of Alox5 was increased 2.5-fold; Alox5 encodes arachidonate 5-lipoxygenase, the key enzyme involved in the biosynthesis of leukotrienes from fatty acids, and the only lipoxygenase that can catalyze the formation of leukotrienes. This enzyme is also required for lipoxinA4 formation, which activates monocytes and macrophages. The mRNA level of Hpdgs, which encodes Author Manuscript Author Manuscript Author Manuscript Author Manuscript Neurobiol Dis. Author manuscript; available in PMC 2016 October 01. Wasseff and Scherer Page 8 prostaglandin D synthase was increased 1.8-fold; this catalyzes the formation of prostaglandin D2, which is mainly produced by oligodendrocytes in the normal CNS but by activated microglia in twitcher mice, which are a genetically authentic model of Krabbe disease. Prostaglandin D2 is a chemoattractant, provides neuroprotection, and may mediate demyelinating in twitcher mice. The mRNA level of Tbxas1, which encodes thromboxane A synthase 1, was increased 3.3-fold; this catalyzes the formation of thromboxane A a powerful inducer of vasoconstriction and platelet aggregation. The mRNA levels of two phospholipases were increased – FD&C Green No. 3 chemical information Pla2g5 and Plcg2. Pla2g5 encodes phospholipase A2 group V, the enzyme that catalyzes the hydrolysis of membrane phospholipids to generate lysophospholipids and free fatty acids, including arachidonic acid. It also induces leuokotrines biosynthesis in neighboring inflammatory cells. Plcg2 encodes the transmembrane signaling enzyme phospholipase C gamma 2, which catalyzes the conversion of 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate to 1Dmyoinositol 1,4,5-trisphosphate and diacylglycerol both important secondary messengers that transmit signals from surface receptors. DAG is well known as a secondary messenger of