ABSTRACT
Objective To establish tree shrew model of type 2 diabetes mellitus (T2DM) combined with cerebral ischemia (IS),and to explore the regulatory mechanism of ischemic postconditioning (PC) on gene differential expression in cerebral cortex under metabolic abnormalities and cerebral ischemia condition.Methods Seventy tree shrews were divided into control,T2DM,IS,T2DM+IS and T2DM +IS +PC groups (n =14 each group).The experimental diabetes model was established by the combined use of high fat diet breeding with streptozocin injection in tree shrew.The local cerebral thrombosis was induced by photochemical reaction in tree shrews,and ischemic PC was established at 4h after cerebral ischemia followed by clipped ipsilateral common carotid artery three times (5 min/time).The metabolic status of tree shrews was measured by serum biochemical markers.TTC staining,HE staining,and electron microscopy were used to observe the changes of the body's metabolic status at 24h after IS.RNA-seq was used to analyze differentially expressed genes.Results The ultrastructure of brain cells was abnormal and the cerebral infarction area was the largest in T2DM+IS tree shrews (P＜0.01).Compared with control group,body weight of tree shrews in T2DM + IS group was significantly reduced (P＜ 0.01) while blood glucose,total cholesterol,low density lipoprotein-cholesterol,triglyceride,and C-reactive protein (CRP) levels were markedly increased(all P＜0.01).The RNA-seq analysis showed that there were 1 629 differentially expressed genes (1 109 up-regulated genes and 520 down-regulated genes) in T2DM + IS group vs control group.However,ischemic PC deceased the cortical infarction area (P＜0.01)and reduced blood glucose,lipid and CRP levels (P＜0.05),with 520 differential expression genes (203 up-regulated genes and 317 down-regulated genes).Conclusion Ischemic PC improves the metabolic disturbance-aggravated ischemic brain injury in T2DM tree shrews,which may be related to its regulation on gene expression in cerebral cortex.
ABSTRACT
Objective To introduce a single person operation procedure for tail vein injection in conscious tree shrews,and to improve the success rate of injection. Methods The tree shrew was fixed by a canvas glove and a clamp. The tail of the tree shrew was fixed by the operator's left hand, and the drug was injected by the right hand with a 1 mL disposable syringe. Results This method had the advantages of simple operation,economy and practicality,good fixation effect,high matching degree of animals and high success rate of drug administration. Conclusions Compared with other methods,this method has obvious advantages such as single person operation, convenient, time-saving and labor-saving. The cost is low and the effect is good,thus significantly improving the success rate of injection.
ABSTRACT
Objective To Establish a loop-mediated isothermal amplification(LAMP)assay for detection of Salmonella in fecal samples of tree shrews, and report the result of preliminary application of this method. Methods LAMP primers were designed and synthesized according to the conserved sequence of Salmonella specific gene invA (invasive protein gene A). To optimize the reaction time and temperature by setting 10 reaction times(24 to 42 min)and temperature(57℃ to 66℃)and tested its specificity and sensitivity. At the same time, a conventional PCR test was performed to verify and compare with the LAMP assay. 91 fecal samples of wild-derived tree shrews were detected by the LAMP assay. Results The experimental condition was confirmed as 62℃ and 34 min. The sensitivity of Salmonella was 3.36×101CFU/mL, which was 10 to 100 times higher than that of conventional PCR assay. In 10 kinds of intestinal bacteria for LAMP amplification,Salmonella enteritidis and Salmonella paratyphi B were positive,the others were negative. Among the 91 samples of tree shrew fecal samples detected by the LAMP assay, the positive detection rate was 20.88%. The LAMP assay can be completed within 40 min, the result can be observed and judged visually by color changes. Conclusions The LAMP assay established in this study is convenient,rapid,sensitive and specific. It can be used as a rapid measure for large-scale detection of Salmonella in feces of tree shrews.
ABSTRACT
AIM:To investigate the regulatory effect of JAK 2-STAT3 signaling pathway on the neuroprotection of ischemic postconditioning (IPoC) in tree shrews, and to explore the mechanisms of cerebral injury deterioration after in-hibiting the JAK2-STAT3 pathway .METHODS:The model of thrombotic cerebral ischemia was induced by photochemical reaction in tree shrews and the IPoC was established at 4 h after ischemia followed by clipping ipsilateral common carotid ar-tery on the ischemia side for 5 min ( 3 times ) .After IPoC and intracerebroventricular injection of AG 490 ( JAK2 inhibi-tor), the changes of cerebral infarction area were detected by TTC staining , and the histological and ultrastructural changes of cortical neurons were observed under light and electron microscopes , respectively .The protein levels of t-STAT3 and p-STAT3 in the cortical tissue were determined by Western blot .RESULTS:The neuronal pycnosis , mitochondrial swelling and vanish of the mitochondrial cristae were found in cortical cortex , and the infarction area was (24.78 ±3.30)%at 24 h after cerebral ischemia .Meanwhile, the phosphorylation level of STAT 3 protein in the cortical tissue was significantly in-creased (P<0.01).The cortical neuronal damage and mitochondrial swelling were decreased after IPoC , the area of cere-bral infarction was significantly reduced to (17.67 ±1.83)%(P<0.01), and the phosphorylation level of STAT3 protein was further increased ( P<0.01 ) .However , the neuronal damage was aggravated , the infarction area was expanded to (23.85 ±2.77)%(P<0.05) after treatment with AG490, and the phosphorylation level of STAT3 protein was also signif-icantly reduced ( P <0.05 ) .CONCLUSION: IPoC may reduce cerebral injury by regulating the phosphorylation of STAT3 protein, and inhibition of JAK2-STAT3 signaling pathway may counteract the cerebral protective effect of IPoC and aggravate brain injury .
ABSTRACT
Objective To observe the changes of Th1 / Th2 inflammatory factors in the aqueous humor of tree shrews with fusarium solani keratitis,as well as to explore the relationship between Thl / Th2 inflammatory factors and inflammatory response in fusarium solani keratitis.Methods Forty healthy tree shrews were randomly divided into experimental group(n =30) and control group (n =10).Fusarium solani was inoculated into sabina culture medium and cultured at 26 ℃ for 7 days,and then the fungal suspension was collected and the density of spores was adjusted to 10 × 109 CFU · mL-1.In the experimental group,50 μL fungal spore suspension was injected into the center of the cornea stroma,while the control group received the same amount of saline.Next the levels of cytokines including interleukin (IL)-1 β,IL-6,IL-4 and IL-10 were analyzed by flow cytometry on day 3,day 7,day 14 after successful modeling,and the changes in types of infiltrating cells were observed by histopathological examination.Results The expression level of IL-1 β and IL-6 (Th1 type cytokines) was the highest on day 7,and the difference was statistically significant at each time point when compared with the control group (all P ＜ 0.05).The expression level of IL-10 (Th2 type cytokines) was the highest on day 14,and the difference was statistically significant at each time point when compared with the control group (all P ＜ 0.05).The difference in IL-4 expression was statistically significant on day 7 (P ＜ 0.05).In addition,histopathological examination showed that the number of infiltration cell reached its peak on day 7,mainly neutrophils,and fungal hyphae were observed to be parallel to the matrix fibers at each time point.Conclusion The proinflammatory cytokines IL-1β and IL-6 and the anti-inflammatory cytokines IL-10 may play an important role in the molecular mechanism of inflammatory response of fusarium solani keratitis in tree shrews.
ABSTRACT
Objective To explore the proliferation characteristics of primary small intestinal epithelial cells of tree shrews and the characteristics of human rotavirus(RV) G1P[8] infection to these cells,and establish a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8].Methods The primary small intestinal epithelial cells were obtained by collagenase Ⅺ and dispase I digestion from tree shrew.After purification and identification,the obtained primary small intestinal epithelial cells were infected with RV.Then,culture supernatants of infected cells were collected every 12 hours after infection.Viral titer and viral load were subsequently determined.Western blot and indirect immunofluorescence observation were used to detect the expression of RV protein VP6 in the primary cells.The infectivity of RV to the tree shrew primary cells was finally evaluated.Results After purification and identification of primary epithelial cells from the tree shrew,high purity above 90% primary tree shrew small intestinal epithelial cells was obtained.These primary small intestinal epithelial cells could be infected with RV virus by comparing the virus infectivity to primary renal cells,HCT116 cells and MA104 cells.The virus titer reached to 2.0×105TCID 50/mL at 72 h after infection.Using Western blot and indirect immunofluorescence observation,the specific viral protein of VP6 was determined to be expressed in the tree shrew primary small intestinal epithelial cells,and were located in the cytoplasm from days 1 to 5.Conclusions The separation,purification and cultivation methods of tree shrew primary small intestinal epithelial cells are successful,and the tree shrew model of RV-infected the tree shrew primary small intestinal epithelial cells is successfully established.
ABSTRACT
Objective To evaluate the effect of Osteoking on blood physiological parameters and organ indexes in the ovariectomized tree shrew model of osteoporotic fracture(OPF). Methods A total of 30 4-month-old female tree shrews(Tupaia belangeri)were randomly divided into control group, model group and treatment group, with 10 in each group. Bilateral ovaries of the tree shrews in the treatment group and model group were removed and osteoporosis was confirmed 6 months later. Then tibial fractures were generated. From the next day of fracture,tree shrews in the treatment group were gavaged with Osteoking once every two days,and tree shrews in the model group and control group were gavaged with equal volume of sterile saline. After gavaging for 3 months,16 blood physiological parameters and 6 organ indexes of the tree shrews in each group were measured. Results Among all of the 16 blood physiological parameters,the number of white blood cells,the absolute value and percentage of lymphocytes(P < 0.01)and monocytes(P < 0.05)in the model group were significantly lower than those in the control group after 3 months. Compared with the model group, the absolute value and percentage of monocytes(P < 0.01)and lymphocytes,the number of white blood cells(P < 0.05) and the spleen coefficient were significantly increased(P < 0.05)in the treatment group. Conclusions Osteoking can improve the immunity of tree shrews with osteoporotic fracture.
ABSTRACT
Objective To analyze the neuroimaging changes of tree shrew models of Alzheimer’ s disease.Methods Nineteen healthy adult female tree shrews were randomly divided into control (5 animals) and model group (14 animals). The model of Alzheimer’s disease was induced by intracerebroventricular injection of Aβ1-40 using a stereotaxic devise and proved successfully by visuospatial congnitive task.The in vivo microstructural changes in the brain of tree shrew AD models and control group (0, 1, 2, 3, 4 weeks) were observed on 1.5T MRI (T2WI), and on 7.0T MRI (12 week)(T2WI, DTI). Results Reference memory errors were increased in the model group at 3 or 4 weeks (P<0.05), and so working memory errors (P<0.05) and period of time to perform (P<0.05, P<0.05, P<0.01) from 2 to 4 weeks.Thus the model was proved to be established successfully.T2WI test and DTI test were carried out.Hippocampus atrophy of the model group at 3 and 4 weeks was observed compared with that at 0 or 1 week or 2 weeks on a 1.5T Philips Gyroscan.Compared with the control group, the temporal horn width in the model group was significantly increased (P<0.01) at 12 weeks on a 7.0T Bruker Biospec Scanner.DTI test at 12 weeks showed that ADC of bilateral hippocampus was up-regulated in the model group ( P<0.01 ) .In the color coded orientation view, loss of the corpus callosum fibers was obvious in the model group. Conclusions Intracerebroventricular injection of Aβ1-40 can lead to learing and memory impairment in tree shrews.There are abnomal MRI signal changes in the brain, and the temporal horn width, hypocampal apparent diffusion coefficient ( ADC) value and corpus callosum damage may provide reference value for the diagnosis of Alzheimer’ s disease.
ABSTRACT
AIM:To assess whether the expression of tight junction (TJ) proteins, occludin/zonula occludins (ZO)-1, and regional cerebral blood flow (rCBF) link to brain edema in tree shrews during thrombotic cerebral ischemia and ischemic postconditioning (PC), and to explore how TJ affects brain edema and cerebral infarction .METHODS:Tree shrews were randomly grouped into control , ischemia and cerebral ischemia +PC (n=23), and the remaining 3 ani-mals were used for magnetic resonance imaging ( MRI) .The local cerebral thrombosis were induced by photochemical reac-tion in the tree shrews , and ischemic PC was established at 4 h after induction of cerebral ischemia followed by clipped ipsi-lateral common carotid artery (5 min ×3).The changes of the neural ultrastructure were observed under electron micro-scope.The neuronal apoptosis was analyzed by the method of TUNEL .Laser Doppler brain flowmetry was used to monitor the rCBF.The protein levels of occludin/ZO-1 were determined by immunochemistry and Western blot .The cerebral in-farction volume was detected by MRI .The brain water content was measured by dry-wet weight method .RESULTS: In-duction of cerebral ischemia led to a significant reduction of the normal neuron numbers in the hippocampal CA 1 area, and conversely, the number of neurons with abnormal ultrastructure was increased .The TUNEL positive cells were increased significantly (P<0.01) in ischemia group.Moreover, the rCBF decreased significantly (P<0.01), and occludin/ZO-1 protein expression decreased ( P<0.01 ) .The brain water content and cerebral infarction volume were significantly in-creased (P<0.01).Ischemic PC increased the rCBF and the occludin/ZO-1 expression, but reduced the brain water con-tent, the TUNEL positive cells, and the infarction volume (P<0.01).CONCLUSION:Ischemic PC increases the rCBF but not the local water content , suggesting that reduced cerebral infarction volume after ischemia PC is associated with the attenuation of cerebral edema by the enhancement of occludin /ZO-1 protein expression .
ABSTRACT
Objective To study the effect of low frequency electrical stimulation on blood hormone levels in differ -ent animals.Methods At 12 h, 24 h and 36 h after low frequency electrical stimulation to Chinese tree shrews , Wistar rats, and BALB/c mice, respectively, the blood noradrenaline (NA) and endothelin (ET) levels were determined by ra-dioimmunoassay ( RIA) .Results The blood NA and ET levels of electrical stimulation group were significantly higher than that in the control group (P Wistar rats >BALB/c mice.Conclusions Low frequency electrical stimulation can promote the release of noradrenaline and en -dothelin in Chinese tree shrews , Wistar rats and BALB/c mice.Reserpine can reduce the stress induced by low frequency electric stimulation to the body .
ABSTRACT
Objective To study the effect of low frequency electrical stimulation on hormone levels in organs of Chinese tree shrews after death.Methods Giving Chinese tree shrews low frequency electrical stimulation.At 0 h, 3 h, 6 h, 12 h, 18 h, 24 h, 36 h and 72 h after death, the thyroid, liver, spleen were taken,and the levels of endothelin (ET), atrial natriuretic factor( ANF) , thromboxane ( TX) were determined by RIA method.At 0 h after death, midbrain ventral tegmental area ( VTA) of Chinese tree shrews was taken to detect the c-fos expression.Results After electrical stimula-tion, ET, ANF, TX levels in the cadaver organs and VTA c-fos expression of Chinese tree shrews were significantly in-creased than in the control group.The contents were decreasing with the time after death.Conclusions Low frequency e-lectrical stimulation can induce the synthesis and release of hormones in organs and c-fos expression in brain tissue of Chi-nese tree shrews.
ABSTRACT
Objective To investigate the expression levels of BDNF, trkB and ChAT mRNA and proteins in the brain of adult tree shrews ( Tupaia belangeri ) .Methods Quantitative real-time PCR was employed to detect the expression levels of BDNF, trkB and ChAT mRNA in the hippocampus, basal ganglia and frontal cortex of adult tree shrews.The expression levels of BDNF, trkB and ChAT proteins andβ-actin was used as internal standard.Results The expression level of BDNF mRNA was highest in the hippocampus of adult tree shrew, and there were significant differences between that in the hippocampus, and basal ganglia and frontal cortex (P0.05) in the expressions of trkB protein among the hippocampus, basal ganglia and frontal cortex of the adult tree shrews.There were no significant differences in expressions of ChAT mRNA and protein among the hippocampus, basal ganglia and frontal cortex in adult tree shrews ( P>0.05 ) . Conclusions The expression levels of ChAT mRNA were consistent with that of ChAT protein in the hippocampus, basal ganglia and frontal cortex of adult tree shrews, while the expression levels of BDNF and trkB mRNA were not consistent with their proteins, which might indicate that the transcriptional regulation pattern might be more complex.Tree shrew is a valuable animal model in the study of mechanism of BDNF/trkB gene expression.
ABSTRACT
Objective To investigate the infection status of Toxoplasma gondii in different colonies of tree shrews and then provide the basis for parasitological monitoring .Methods Each of the forty blood samples were randomly collected from three tree shrews colonies : wild origin, domesticated and first generation, respectively.Both indirect hemagglutination test (IHA) and PCR assay were used to detect the Toxoplasma gondii.Results No positive sample of Toxoplasma gondii was detected from either IHA or PCR results .The results from IHA and PCR assays were in coincidence with each other.Conclusions According to the survey none of the tree shrews from the three groups is infected with Toxoplasma gondii.More samples or infection experiments are needed to determine whether tree shrews can be infected with Toxoplasma gondii.
ABSTRACT
Objective To understand the histological characteristics of the major endocrine organs of tree shrew , and provide a normal histological atlas of endocrine organs of tree shrew .Methods Ten artificially fed healthy tree shrews were killed and dissected after anesthesia .The thyroid, parathyroid, adrenal and pituitary glands were observed by gross inspection and samples were taken for routine histological examination with HE staining .Results ( 1 ) The thyroid gland was pale yellow, located on both sides of the 2-4 tracheal rings.The thyroid gland was plate-shaped, its surface was covered with a thin fibrous capsule . The thyroid parenchyma was divided into several lobules by stretched capsule membrane .Follicular and parafollicular cells were distributed in the lobules , and red colloid was present in follicular cavity.(2) Each side had one parathyroid , located on the cranial or the outer surface of the middle part of the thyroid gland, and was slightly covered by thyroid .The gland was round or oval , and its parenchyma was made up of the principal cells and eosinophil cells , and acinar structure appeared in the parenchyma .( 3 ) The adrenal glands were oval , yellow color, located in the renal hili , and linked to the kidneys .They were surrounded by a thin capsule .The parenchyma was divided into cortex and medulla .The cortex was divided into zona glomerulosa , zona fasciculata and zona reticularis from outside to inside.The zona glomerulosa was the thickest layer and the zona fasciculata was the thinnest .The medulla cells formed clumps or mesh, with central vein in the central part .(4) The pituitary gland was located in the sella turcica , with no recessus hypophysis .The pituitary gland was composed of the adenohypophysis and neurohypophysis .Its surface was covered with a connective tissue capsule .The pituitary gland was divided into distal part , middle part and pars tuberalis . neurohypophysis was made up of neural and pars infundibularis .Conclusions The histological atlas of endocrine organs in the tree shrew is established , which is close to that of the primate animals in the morphology , and provide histological evidence for the study of tree shrew endocrine organs and disorders , as well as the animal model of human diseases .
ABSTRACT
AIM:To explore the changes and significance of Kupffer cells in the process of tree shrew chroni -cally infected with hepatitis B virus (HBV).METHODS:The animals were divided into 3 groups.Group A consists of 6 tree shrews that were identified as persistently infected with HBV;group B consists of 3 tree shrews that were suspected as persistently infected with HBV;group C consists of 4 tree shrews that were not inoculated with HBV and were applied as normal controls.Liver biopsies were collected regularly from all animals , and the Kupffer cells were isolated , purified and primarily cultured.The techniques of flow cytometry , immunohistochemistry, lysosomal fluorescent probe staining and real-time RT-PCR were applied to determine the number and function of these Kupffer cells .RESULTS: The result showed that the count and proportion of CD 163+cells in group A were significantly higher than those in group B and group C ( P<0.05).Meanwhile, the fluorescence intensity levels of lysosomal , the number of lysozyme-positive cells and the mRNA ex-pression level of TNF-αin the Kupffer cells in group A were significantly lower than those in group B and group C ( P<0.05).CONCLUSION:Kupffer cells may play a regulatory role during host’s chronic HBV infection.
ABSTRACT
Viral hepatitis is a major liver disease caused by virus infection .Viral hepatitis is popular in China , mainly caused by hepatitis B and hepatitis C viruses .Experimental animal model is a necessary platform for the research on mechanism of viral infection and pathogenicity , for treatment and vaccine development .Up to date, a great progress in the development of viral hepatitis animal models has been achieved in spite of the most of findings are limited to hepatitis B and C.Here, we summarize the recent findings of viral hepatitis animal models , focusing on the tree shrew animal model and its modeling strategy .
ABSTRACT
AIM: To observe the changes of VEGF expression in different subfield of brain in tree shrews during hyperglycemia and focal cerebral ischemia, in order to explore the relationship between cerebral ischemia, hyperglycemia and VEGF. METHODS: High blood glucose in tree shrews was induced by intraperitoneal injection of streptozotoctin. Focal cortical thrombotic cerebral ischemia was induced by photochemical method in tree shrews. At 4 h, 24 h and 72 h after cerebral ischemia, the histopathological changes and hippocampal neuronal density were examined. VEGF expressions in the ischemic core, penumbra and contralateral cerebral cortex were detected by immunohistochemistry technique at different times after cerebral ischemia. RESULTS: The results of histopathological study showed that there was infarction zone in the exposured cerebral cortex at 4 h after photochemical reaction, and the damage was most severe at 24 h, subsequently accompanied with the glia multiplication and rehab reaction at 72 h. The animals in hyperglycemic ischemic group suffered from greater neurological lesion than the normoglycemic stroke animals, especially at 24 h (P<0.01) and 72 h (P<0.05) after cerebral ischemia. Immunohistochemical analyses of VEGF expression revealed that it started to increase at 4 h after brain ischemia in the penumbra, reached a peak at 24 h, and weakened at 72 h. The stimulated VEGF production was also observed in hyperglycemic only group. When hyperglycemia and brain ischemia were combined, the VEGF expression was higher than that in hyperglycemic only group (P<0.05). Compared to normoglycemic ischemic group, no additivity of the effects of hyperglycemia combined with brain ischemia was observed. CONCLUSION: (1) The model of experimental hyperglycemia and cerebral ischemia is replicated successfully by applying the method combined in vivo injection of streptozotocin in the lower primate tree shrew with thrombotic focal cerebral ischemia. (2) This study shows that hyperglycemia aggravates the focal cerebral ischemia damage. (3) Cerebral ischemia and hyperglycemia both can independently up-regulate VEGF expression, but there is no additional increase in VEGF expression when hyperglycemia combined with brain ischemia is applied.
ABSTRACT
AIM:To study the effects of ischemic postconditioning(PC) on regional cerebral blood flow(rCBF) and astrocyte(AS) activation in hippocampus CA1 area and to explore the possible mechanism of ischemic PC affecting glial fibrillary acidic protein(GFAP) expression during focal cerebral thrombosis.METHODS:The thrombotic focal cerebral ischemia was induced by photochemical reaction in tree shrews,and ischemic postconditioning was established by cliped ipsilateral carotid of the animal at 4 h after cerebral ischemia.The rCBF and GFAP expressions in hippocampus CA1 area were detected,respectively,by laser-Doppler(LD) fowmeter and immunohistochemistry.RESULTS:The numbers of GFAP positive cells were increased markedly and GFAP expression enhanced(P
ABSTRACT
AIM: To observe the changes of glutamate and calcium within the hippocampal microenvironment in mitochondrial stress.METHODS: A lateral hippocampus was microperfused with glutamate and calcium chloride solution by a kind of single-pumped push-pull perfusion system in Tree Shrews.At 24 h,the expression of cytochrome C(Cyt C)was observed by immunochemistry.Also,the hippocampus was removed,then mitochondria and cytoplasmic fragment were divided by low temperature centrifugation and the distribution of cytochrome C was assessed through Western blotting.The relative amounts of caspase-3 and caspase-9 mRNA were evaluated by real time fluorescence polymerase chain reaction.In the treated group,cyclosporin A(CsA,40 mg/kg) was intravascularly injected at 6 h after perfusion of glutamate-calcium chloride solutions into the hippocampus and inspected the above-mentioned items at 24 h.RESULTS: In the glutamate-calcium group,compared with the control group,cytochrome C immunoreactivity increased and the content of hippocampal mitochondrial cytochrome C decreased.Also,the cytochrome C was detected in cytosol.Cyclosporin A treatment at 6 h after microperfusion,the cytochrome C expression weakened and no Cyt C in cytosol fraction was observed.By real time PCR,in relation to the control group,the caspase-3 and caspase-9 mRNA was higher in the glutamate-calcium group.Cyclosporin A treatment cut down both caspase-3 and caspase-9 mRNA contents.CONCLUSION: The accumulation of glutamate and calcium may promote Cyt C release,caspase cascade activation and the mitochondrial stress.The neuroprotection of CsA may results from uniquely inhibiting the mitochondrial permeability transition pore,and preventing Cyt C release and caspase activation.