Jian-Ping Zhang

Free Fatty Acids, Neutral Glycerides, and Phosphoglycerides in Transient Focal Cerebral Ischemia

Abstract: Cerebral ischemia is known to cause an increase in levels of free fatty acids (FFAs) and diacylglycerols (DGs), although the mechanism(s) leading to these changes is not well understood. In this study, we examined FFA and DG levels along with those of other lipids in rats during and after transient focal cerebral ischemia induced by temporary occlusion of the right middle cerebral artery (MCA) and both common carotid arteries. During the duration of ischemia (15–60 min), there was a time‐dependent increase (two‐ to 10‐fold) in FFA levels in the right MCA cortex, whereas levels of DG and other lipids were not altered appreciably. FFA levels in right MCA cortex returned to near control values after reperfusion. However, following a 60‐min ischemic insult, there was a second phase of FFA level increase that was evident after 16 h. The FFAs accumulated during the ischemia period were different from those after reperfusion, suggesting differences in mechanisms for their release. During the second phase of FFA release, there were increases in levels of DGs and triacylglycerols (TGs) with unusually high proportions of 20:4(n‐6) and 22:6(n‐3). The increases in FFA, DG, and TG levels were marked by a decrease in content of phosphoglycerides (PGs). It is interesting that the increases in levels of FFAs and neutral glycerides accounted only for 10% of the total PGs depleted. The lipid changes during this reperfusion period correlated well with the development of cortical infarct. Because FFAs are potent inhibitors of mitochondrial respiratory function, the time‐dependent FFA accumulation during the ischemia period may be an important determinant for the extent of ischemia‐induced injury after reperfusion.

In situ hybridization of rnRNA expression for IP3 receptor and IP3-3-kinase in rat brain after transient focal cerebral ischemia

Abstract Loss of intracellular calcium homeostasis has been regarded an important factor underlying neuron cell death after cerebral ischemic insult. In the brain, a major mechanism for regulation of intracellular calcium is through the signal transduction pathway involving hydrolysis of poly -phosphoinositides and release of the second messenger, inositol 1,4,5-trisphosphate (IP3). IP3 mobilizes calcium by interacting with an intracellular receptor. Upon its release after agonist stimulation, this second messenger is catabolized by a 3-kinase and a 5-phosphatase. In this study, in situ hybridization was carried out to examine the mRNA expression of IP3 receptor (IP3R) and IP3 3-kinase (IP3K) in rat brain cortex after transient focal cerebral ischemia induced by temporary occlusion of the middle cerebral artery (MCA) and the common carotid arteries (CCAs). Results indicate a large decrease (52%) in IP3R mRNA levels in the ischemic cortex as compared to that in the contralateral side at 4 h after a 45 min ischemic insult. By 16 h, practically no IP3R mRNA could be detected in the ischemic cortex. On the other hand, IP3K mRNA levels remained unaltered until 16 h after reperfusion, during which time, expression in the infarct core decreased but that surrounding the core area increased instead. Hybridization of adjacent brain sections with probes for neuron specific enolase (NSE) and β-actin indicated also a time-dependent decrease in mRNA levels after ischemia, but these changes were less dramatic as compared to IP3R. At 16 and 24 h after reperfusion, there was an increase in β-actin mRNA in cortical areas outside the MCA cortex, suggesting of reactive gliosis. The exquisite sensitivity of IP3R gene expression to focal cerebral ischemic insult and its perturbation during the early reperfusion period suggests an important role of the IP3R in cerebral ischemic injury.

Metabolism of inositol 1,4,5-trisphosphate in mouse brain due to decapitation ischemic insult : effects of acute lithium administration and temporal relationship to diacylglycerols free fatty acids and energy metabolites

Previous studies have shown that global cerebral ischemia induced by decapitation leads to the stimulated hydrolysis of poly-phosphoinositides. In this study, the decapitation model was used to further examine the temporal events related to metabolism of Ins(1,4,5)P3 and the release of diacylglycerols (DGs) and free fatty acids (FFAs) in the mouse brain. Since lithium administration is known to inhibit inositol monophosphatase activity in brain, the effects of acute lithium injection on Ins(1,4,5)P3 metabolism were also examined. Cerebral ischemia induced by decapitation of C57 Bl/6J mice resulted in transient increases of Ins(1,4,5)P3, Ins(1,4)P2 and Ins(4)P which peaked at 35, 65 and 125 s, respectively. The level of Ins(1)P, however, was not altered. Mice administered lithium by intraperitoneal injection (8 meq/kg for 4 h) gave rise to a 40- and 4-fold increase in levels of Ins(1)P, Ins(4)P, respectively, a 20% increase in levels of Ins(1,4)P2 but no apparent changes in the levels of Ins(1,4,5)P3. Decapitation also induced an increase in the levels of DGs and FFAs. Unlike the transient appearance of Ins(1,4,5)P3, however, DG levels increased steadily for 2 min and then reached a plateau whereas the FFAs showed a lag time of 35 s prior to a biphasic increase. During the initial 2 min after decapitation, there was a preferential increase in the DG species containing 18:0 and 20:4. Lithium administration did not alter the decapitation-induced release of DG and FFA. As expected, decapitation gave rise to a rapid decrease in the levels of phosphocreatine and ATP and the decline in ATP was marked by a transient appearance of ADP and a concomitant increase in AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ischemic tolerance on mRNA levels of IP3R1, β-actin, and neuron-specific enolase in hippocampal CA1 area of the gerbil brain

Global cerebral ischemia induced to Mongolian gerbils by ligation of common carotid arteries (CCAs) is known to result in injury to the hippocampal CA1 region. In this study, we examined whether neuronal injury can be depicted by measuring levels of mRNA encoding inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), neuron specific enolase (NSE) and β-actin and whether these measurements can be use to assess ischemic tolerance. Gerbils were subjected either to cerebral ischemia induced by ligation of both CCAs for 5 min, or to an ischemic tolerance paradigm in which a 2 min ischemic preconditioning was performed 24 hr prior to the 5 min ischemia. At 48 hr after the 5 min ischemic insult, significant decreases in mRNA levels for IP3R1 (26%), NSE (38%) and β-actin (50%) could be observed in the hippocampal CA1 region. Although levels of mRNA in the preconditioning group were decreased as compared to the sham control, the levels were significantly higher than those in the ischemic group. These results indicate the feasibility of using mRNA measurement as a parameter to assess cerebral ischemic damage. In addition, based on the differences in the decline in mRNA levels between the ischemia group and the preconditioned ischemia group, it can be concluded that this ischemic tolerance paradigm could offer partial protection (around 45%) against the injury due to the 5 min cerebral ischemic insult.

Regulation of FFA by the acyltransferase pathway in focal cerebral ischemia-reperfusion

Cerebral insult is associated with a rapid increase in free fatty acids (FFA) and arachidonic acid release has been linked to the increase in eicosanoid biosynthesis. In transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, there is an inverse relationship between the increase in FFA and the decrease in ATP, both during the ischemia period and at later time periods after reperfusion. In this study, the focal cerebral ischemia model was used to examine incorporation of [14C]arachidonic acid into the glycerolipids in rat MCA cortex at different reperfusion times after a 60 min ischemia. The label was injected intracerebrally into left and right MCA cortex 1 hr prior to decapitation. Labeled arachidonic acid was incorporated into phosphatidylcholine, phosphatidylethanolamine and neutral glycerides. With increasing time (4–16 hr) after a 60 min ischemia, an inhibition of labeled arachidonate uptake could be found in the right ischemic MCA cortex, whereas the distribution of radioactivity among the major phospholipids was not altered. When compared to labeled PC, there was a 3–4 fold increase in incorporation of label into phosphatidic acid and triacylglycerols (TG) in the right MCA cortex, suggesting of an increase in de novo biosynthesis of TG. In an in vitro assay system, synaptosomal membranes isolated from MCA cortex 8 and 16 hr after a 60 min ischemia showed a significant decrease in arachidonoyl transfer to lysophospholipids, due mainly to a decrease in lysophospholipid:acylCoA acyltransferase activity. Assay of phospholipase A2 activity with both syaptosomes and cytosol, however, did not show differences between left and right MCA cortex or with time after reperfusion. These results suggest that besides ATP availability, the decrease in acyltransferase activity may also contribute to the increase in FFA in cerebral ischemia-reperfusion.

Changes in mRNA levels for group I metabotropic glutamate receptors following in utero hypoxia–ischemia

The expression of group I metabotropic glutamate receptors (mGluR1 and mGluR5) and inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) mRNA was studied by in situ hybridization in the developing rat hippocampus after in utero hypoxia-ischemia. In utero hypoxia-ischemia was induced by clamping the uterine blood vessels of near-term fetuses for 10 min. Fetuses were delivered surgically, resuscitated and raised by foster mothers until postnatal day 7 and 14. Results indicated a temporal delay in the expression of mGluR1 mRNA in the dentate gyrus of the ischemic animals. The mGluR1 mRNA level was significantly lower in the ischemic animals at postnatal day 7, but reached a similar level as that of controls at postnatal day 14. In utero hypoxia-ischemia did not change the temporal-spatial expression pattern of either mGluR5 or IP3R1 mRNA in the hippocampus. Between postnatal day 7 and 14, mGluR5 mRNA showed a high and relatively constant expression, whereas IP3R1 mRNA levels were increased in all regions examined. The differences in the expressions of group I mGluRs indicate that these receptors may have different functions during hippocampal development and may play different roles in excitotoxicity.

Effects of Acute and Chronic Ethanol Administration on the Poly-Phosphoinositide Signaling Activity in Brain

Ethanol ingestion is known to exert diverse physiological effects on many body organs including the central nervous system (CNS). While excess acute ethanol intake can result in sedation, chronic ingestion is associated with development of tolerance and physical dependence and in tum, this may lead to the manifestation of withdrawal hyperexcitability. The biochemical mechanisms underlying the effects of ethanol are not well understood although alterations of many cell surface processes including membrane transport enzymes, receptors and ion channel activities have been implicated (see review by Deitrich et aI., 1989). Some of these effects may be due partly to ethanols ability to disorder membrane lipids. resulting in an alteration of the intricate relationship between proteins and lipids within the membrane. However, it is clear that ethanol does not act globally on the membrane lipids, rather, changes are attributed to the effects of ethanol on specific types oflipids present in different membrane domains (Wood and Schroeder, 1988). In order to better understand the physiological manifestations related to acute and chronic ethanol ingestion, it is important first to identify the ethanol-sensitive biochemical mechanisms in brain and then followed by attempts to understand how adaptation to the changes is developed. Despite some discrepancies due mainly to the methods used for alcohol adminstration, procedures for analysis, age and species of the animal model, many studies have successfully related changes in membrane phospholipids and their fatty acids to the effects of chronic ethanol administration (see review by Sun and Sun, 1985). It appears that acidic phospholipids bearing negative charges are particularly sensitive to the effects of ethanol (Sun et aI., 1987ab; 1989). This notion is supported by studies from our laboratory indicating that chronic ethanol administration resulted in an increase in the levels of acidic phospholipids such as phosphatidylserine (PS) and phosphatidylinositol (PI) in brain synaptic membranes (Sun and Sun, 1983; Sun et aI., 1984). Studies in

The cholinergic receptor-linked phosphoinositide metabolism in mouse cerebrum and cerebellum in vivo.

The cholinergic receptor-linked poly-phosphoinositide hydrolysis was studied in mouse cerebrum and cerebellum after prelabeling the brain with [3H]inositol. I.p. injection of Li (8 meq/kg) to C57Bl/6J mice for 4 h resulted in 14- and five-fold increases in [3H]inositol-labeled inositol monophosphate (IP1) in cerebrum and cerebellum, respectively. The labeled inositol bisphosphate (IP2) was also increased 83 and 19% in cerebrum and cerebellum, respectively. Prior injection of atropine (100 mg/kg) resulted in inhibition of Li-induced increases in labeled IP1 by 74 and 56% in cerebrum and cerebellum, respectively. Administration of pilocarpine (20 mg/kg) to the Li-treated mice for 30 min resulted in further increases in labeled IP1 and IP2 and a concomitant decrease in labeled inositol in cerebrum but not in cerebellum. Mass measurements of IP1 and IP2 isomers by HPLC revealed that inositol 1-monophosphate (Ins(1)P), inositol 4-monophosphate (Ins(4)P) and inositol 1,4-bisphosphate (Ins(1,4)P2) were all increased by pilocarpine administration in the Li-treated mouse cerebrum. The effects of pilocarpine administration in mouse cerebrum (increases in IP1 and IP2) could be completely inhibited by preinjection of atropine. Atropine injection also decreased the levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Surprisingly, a decrease in Ins(1,4,5)P3 level was also found in non-Li-treated mice after pilocarpine administration (30 mg/kg, 10-40 min). Except for the increase (20%) in [32P]-labeled PIP in the cerebrum, Li or Li together with pilocarpine administration did not alter the levels of [3H]inositol or [32P]phosphate-labeled phosphoinositides.(ABSTRACT TRUNCATED AT 250 WORDS)

In utero hypoxic ischemia decreases the cholinergic agonist-stimulated poly-phosphoinositide turnover in the developing rat brain

Perinatal hypoxic-ischemic (HI) insult is known to cause cellular and molecular disturbances leading to functional and behavioral abnormalities during brain development. In this study, we examined the effects of an in utero HI insult on poly-phosphoinositide turnover in vivo in the cerebrum and cerebellum as well as cholinergic-stimulated turnover in cortical slices from developing rat brain. In utero HI treatment was carried out by clamping the uterine blood vessels of near-term fetuses for 5, 10 and 15 min followed by resuscitation of the newborn pups. The in vivo protocol for examining poly-PI signaling activity in 2 week-old pup brain involved intracerebral injection of [3H]inositol for 16 hr and subsequent intraperitoneal injection with lithium (8 meq/kg) for 4 hr prior to decapitation. In the control pups, lithium elicited a 2.6 fold increase in labeled inositol phosphate (IP) in the cerebrum as compared to a 1.3 fold increase in the cerebellum. In utero HI insult (5 to 15 min) resulted in a small increase in labeled IP in the cerebrum but not in the cerebellum. Carbachol stimulation of poly-PI turnover was examined in brain slices prelabeled with [3H]inositol in vivo. Incubation of the prelabeled slices with carbachol in the presence of LiCl (10 mM) resulted in a time-, dose- and age-dependent increase in labeled IP. Brain slices from 2 week-old pups that experienced in utero HI-treatment for 10 and 15 min (but not 5 min) showed a significant decrease in carbachol-stimulation of labeled IP as compared with control pups. These results indicate the effects of in utero HI on the choninergic-stimulated poly-PI signaling pathway and its implication on related functional deficits in the developing brain.