Hese and our own observations illustrate that monitoring body temperature during the procedure is of particular importance, as the body temperature of the animal needs to remain jasp.12117 stable during ischemia, preferably up to the moment the animal regains consciousness. With regard to adequate temperature control, it is important to keep in mind that the body temperature of animals, sedated at room temperature can decrease several degrees following administration of ketamine and xylazine [44]. Also, heating the animal with a heat lamp and heating mat needs to be closely monitored as body temperature can spike at 38 or higher, increasing the variability within the group. As an alternative, a neonatal incubator provides a more stable temperature controlled environment which avoids body temperature spikes and prevents the anaesthesia-associated temperature drop when animals are put inside immediately after sedation (own observations). Overall, it should be noted that temperature settings depend on the lab environment, the mouse strain, and mouse weight (as fat tissue can insulate) and therefore require an empiric optimization and standardization.Experimental evaluation of unilateral ischemia-reperfusion in miceEffect of body temperature during ischemia on fibrotic outcome. Unilateral renal ischemia-reperfusion injury (UIRI) results in a significant reduction of renal mass (p<0.05) at all temperature conditions tested. As depicted in Fig 1A, UIRI at 37 caused a ?5 reduction (p<0.05) in renal mass, whereas the mildest temperature condition tested, i.e. 34 , also caused a less pronounced (p<0.05) but still severe reduction in renal mass (?0 scan/nsw074 ). Masson’s stain showed prominent renal damage and severe loss of structure, atrophic renal cortex with disruption of tubular architecture, marked tubule necrosis and intratubular casts, and extensive interstitial inflammatory infiltration (Fig 2A). Quantification of fibrosis by AG-490 site collagen I immunostaining demonstrated an increased deposition of collagen I for all body buy MK-8742 temperatures under study as compared to sham (p<0.05), with a more pronounced increase in collagen I staining for UIRI at 37 as compared to the lower body temperatures (35 and 34 ) (p<0.05) (Fig 3C). As shown in Fig 4, 12 weeks after UIRI a significant increase in gene expression of fibrosis-related genes Col I, TGF, CCN2 and CCN3 was observed in renal cortex tissue in all core body temperature conditions tested as compared to sham (p<0.05). The long-term UIRIinduced expression of these genes is also temperature-dependent: higher expression with higher temperature during ischemia (37 and 36 vs. 35 and 34 ; p<0.05) (Fig 4). Effect of body temperature during ischemia on long-term expression of inflammatory and tubular injury markers. Analysis of gene expression of hepatitis A virus receptorPLOS ONE | DOI:10.1371/journal.pone.0152153 March 23,8 /An Ischemic Mouse Model for AKI to CKDFig 1. Ischemic kidney weight at euthanasia. Kidney weights are corrected for body weight. *: p<0.05, ? p<0.05 vs. Sham. A: UIRI was performed for 30 minutes at 37 (n = 5), 36 (n = 4), 35 (n = 10) or 34 (n = 5) and animals were euthanized 12 weeks after UIRI. UIRI results in a significant reduction of renal mass (p<0.05) at all temperature conditions tested. B: UIRI was performed for 30, 21 or 18 minutes at 36 and animals were euthanized 6 weeks (resp. n = 5, n = 12, n = 6) and 12 weeks (resp. n = 4, n = 5, n = 10) after UIRI. UIRI causes an ischemia time-depen.Hese and our own observations illustrate that monitoring body temperature during the procedure is of particular importance, as the body temperature of the animal needs to remain jasp.12117 stable during ischemia, preferably up to the moment the animal regains consciousness. With regard to adequate temperature control, it is important to keep in mind that the body temperature of animals, sedated at room temperature can decrease several degrees following administration of ketamine and xylazine [44]. Also, heating the animal with a heat lamp and heating mat needs to be closely monitored as body temperature can spike at 38 or higher, increasing the variability within the group. As an alternative, a neonatal incubator provides a more stable temperature controlled environment which avoids body temperature spikes and prevents the anaesthesia-associated temperature drop when animals are put inside immediately after sedation (own observations). Overall, it should be noted that temperature settings depend on the lab environment, the mouse strain, and mouse weight (as fat tissue can insulate) and therefore require an empiric optimization and standardization.Experimental evaluation of unilateral ischemia-reperfusion in miceEffect of body temperature during ischemia on fibrotic outcome. Unilateral renal ischemia-reperfusion injury (UIRI) results in a significant reduction of renal mass (p<0.05) at all temperature conditions tested. As depicted in Fig 1A, UIRI at 37 caused a ?5 reduction (p<0.05) in renal mass, whereas the mildest temperature condition tested, i.e. 34 , also caused a less pronounced (p<0.05) but still severe reduction in renal mass (?0 scan/nsw074 ). Masson’s stain showed prominent renal damage and severe loss of structure, atrophic renal cortex with disruption of tubular architecture, marked tubule necrosis and intratubular casts, and extensive interstitial inflammatory infiltration (Fig 2A). Quantification of fibrosis by collagen I immunostaining demonstrated an increased deposition of collagen I for all body temperatures under study as compared to sham (p<0.05), with a more pronounced increase in collagen I staining for UIRI at 37 as compared to the lower body temperatures (35 and 34 ) (p<0.05) (Fig 3C). As shown in Fig 4, 12 weeks after UIRI a significant increase in gene expression of fibrosis-related genes Col I, TGF, CCN2 and CCN3 was observed in renal cortex tissue in all core body temperature conditions tested as compared to sham (p<0.05). The long-term UIRIinduced expression of these genes is also temperature-dependent: higher expression with higher temperature during ischemia (37 and 36 vs. 35 and 34 ; p<0.05) (Fig 4). Effect of body temperature during ischemia on long-term expression of inflammatory and tubular injury markers. Analysis of gene expression of hepatitis A virus receptorPLOS ONE | DOI:10.1371/journal.pone.0152153 March 23,8 /An Ischemic Mouse Model for AKI to CKDFig 1. Ischemic kidney weight at euthanasia. Kidney weights are corrected for body weight. *: p<0.05, ? p<0.05 vs. Sham. A: UIRI was performed for 30 minutes at 37 (n = 5), 36 (n = 4), 35 (n = 10) or 34 (n = 5) and animals were euthanized 12 weeks after UIRI. UIRI results in a significant reduction of renal mass (p<0.05) at all temperature conditions tested. B: UIRI was performed for 30, 21 or 18 minutes at 36 and animals were euthanized 6 weeks (resp. n = 5, n = 12, n = 6) and 12 weeks (resp. n = 4, n = 5, n = 10) after UIRI. UIRI causes an ischemia time-depen.