Fig 1: Effects of systemic interventions on hepatic neurotransmitters and damage in donors with severe steatosis or alcoholic liver disease. (A) Experimental Protocol. Diagrammatic representation of the experimental protocol based on ACh and 5-HT administration (iv). Control rats and rats with either severe steatosis or ALD underwent either Sham or BD surgical procedures. Immediately after BD induction, animals received intravenous administration of either ACh or 5-HT via the infrahepatic vein. After 8 h of BD induction or after 8 h under anesthesia in the case of the Sham-operated animals, plasma and liver tissue samples were collected for the biochemical and histological analyses shown in the figure. (B) Effects of systemic interventions on ACh and 5-HT levels in liver grafts prior to retrieval from donors. The sample size was n = 6 rats per group and for each measurement. (C) Effects of systemic interventions on damage in liver grafts prior to retrieval from donors. Transaminases and bilirubin were measured in plasma as markers of hepatic injury. Caspase-3 activity was measured in tissue as a marker of apoptosis. Histological liver damage was evaluated with H&E staining and scored; representative images of liver sections stained with H&E at 10X (scale bars correspond to 100 µm) and TUNEL staining and the percentage of apoptotic cells, were used to assess apoptosis in liver tissue. Representative TUNEL-stained liver sections at 20X (scale bars correspond to 100 µm) are shown. The sample size was n = 6 rats per group and for each measurement. (D) Effects of systemic interventions on regenerative failure in liver grafts prior to retrieval from donors. PCNA, HGF, BDNF, CNTF and TGF-β in liver tissue were analyzed as indicators of hepatic regeneration. The sample size was n = 6 rats per group and for each measurement. ALD alcoholic liver disease, BD brain death, ACh acetylcholine, iv intravenous, 5-HT serotonin, H&E hematoxylin & eosin, TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling, PCNA proliferating cell nuclear antigen, HGF hepatocyte growth factor, BDNF brain-derived neurotrophic factor, CNTF ciliary neurotrophic factor, TGF-β transforming growth factor β. *P < 0.05 versus Sham; + P < 0.05 versus BD.
Fig 2: Effects of brain death and therapeutic interventions on hepatic neurotransmitters and damage in liver grafts with severe steatosis or alcoholic liver disease after ex vivo normothermic reperfusion. (A) Experimental Protocol. Diagrammatic representation of the experimental protocol based on IPNS electrical stimulation, ACh(ma) or ACh(iv) and 5-HT(iv). Control, severe steatosis, and ALD rats underwent Sham or BD procedures. Immediately after BD induction, IPNS stimulation, ACh (ma), ACh (iv), or 5-HT (iv) was administered via the infrahepatic vein. After 8 h of BD or anesthesia (Sham), liver grafts were flushed and preserved in UW solution, subjected to CI at 4 °C for 24 h, and reperfused ex vivo via the portal vein for 2 h at 37 °C using a recirculating system. Perfusate and liver tissue were collected for analyses shown in the figure. (B) Effects of intestinal and systemic interventions on hepatic norepinephrine, ACh and 5-HT levels after 24 h of cold ischemia and 2 h of ex vivo reperfusion. The sample size was n = 6 rats per group and for each measurement. (C) Effects of intestinal and systemic interventions on heaptic damage and regeneration after 24 h of cold ischemia and 2 h of ex vivo reperfusion. Transaminase (hepatic injury) were measured in the perfusate and MDA (oxidative stress) in liver. Histological liver damage was assessed and scored using H&E-stained sections; representative images (10 ×) are shown. Sinusoidal inflammation could not be fully evaluated due to the absence of erythrocytes and inflammatory cells. Hepatic PCNA, HGF, BDNF, CNTF, and TGF-β were analyzed as regenerative markers. The sample size was n = 6 rats per group and for each measurement. ALD alcoholic liver disease, BD brain death, IPNS stimulation electrical stimulation of intestinal parasympathetic nervous system, ACh acetylcholine, iv intravenous, ma mesenteric artery, 5-HT serotonin, UW University of Wisconsin, CI cold ischemia, MDA malondialdehyde, H&E hematoxylin & eosin, PCNA proliferating cell nuclear antigen, HGF hepatocyte growth factor, BDNF brain-derived neurotrophic factor, CNTF ciliary neurotrophic factor, TGF-β transforming growth factor β. *P < 0.05 versus Sham; +P < 0.05 versus BD.
Fig 3: Effects of intestinal interventions on liver injury, apoptosis and regeneration in donors with severe steatosis or alcoholic liver disease. (A) Experimental Protocol. Diagrammatic representation of experimental protocol based on IPNS electrical stimulation and ACh administration (ma). Control, severe steatosis, and ALD undergoing Sham or BD surgical procedures received either IPNS electrical stimulation or ACh (ma) immediately after BD induction. After 8 h of BD induction, or after 8 h under anesthesia (Sham-operated animals), livers were collected for biochemical and immunohistochemical analyses shown in the figure. (B) Effects of intestinal interventions on ACh and 5-HT in liver grafts prior to retrieval from donors. The sample size was n = 6 rats per group and for each measurement. (C) Effects of intestinal interventions on damage in liver grafts prior to retrieval from donors. Transaminases and bilirubin were measured in plasma (markers of hepatic injury). MPO and MDA were determined in liver as indicators of inflammation (MPO reflecting neutrophil accumulation, and MDA indicating oxidative stress) Caspase-3 activity measured in liver, indicating apoptosis. Histological liver damage was evaluated with H&E staining and scored; representative images of liver sections stained with H&E at 10X (scale bars correspond to 100 µm) and TUNEL staining and the percentage of apoptotic cells, were used to assess liver apoptosis. Representative TUNEL-stained liver sections at 20X are shown. The sample size was n = 6 rats per group and for each measurement. (D) Effects of intestinal interventions on regenerative failure in liver grafts prior to retrieval from donors. PCNA, HGF, BDNF, CNTF and TGF-β in liver were analyzed as indicators of regeneration. The sample size was n = 6 rats per group and for each measurement. ALD alcohol liver disease, BD brain death, IPNS stimulation electrical stimulation of the intestinal parasympathetic nervous system, ACh acetylcholine, ma mesenteric artery, 5-HT serotonin, MPO myeloperoxidase, MDA malondialdehyde, H&E hematoxylin & eosin, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, PCNA Proliferating cell nuclear antigen, HGF hepatocyte growth factor, BDNF brain-derived neurotrophic factor, CNTF ciliary neurotrophic factor, TGF-β transforming growth factor β. *P < 0.05 versus Sham; +P < 0.05 versus BD.
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