A. Muralikrishna Rao

The biphasic opening of the blood-brain barrier in the cortex and hippocampus after traumatic brain injury in rats

This study examined the time course of the blood-brain barrier (BBB) opening and correlated this with brain edema formation after a lateral controlled cortical impact (CCI) brain injury in rats. Quantitative measurement of Evans blue (EB) extravasation using fluorescence was employed at 2, 4, 6 h and 1, 2, 3, 4 and 7 days after injury. Brain edema was measured by specific gravity of the tissue at corresponding time points. Two prominent EB extravasations were observed at 4-6 h and 3-day after injury in the injury-site cortex and the ipsilateral hippocampus. Brain edema became progressively more severe over time and peaked at 24 h after injury and began to decline after day 3. These results suggest that there is a biphasic opening of the BBB after CCI brain injury and the second opening of the BBB does not contribute to a further increase in edema formation.

Lipid alterations in transient forebrain ischemia: possible new mechanisms of CDP-choline neuroprotection.

Abstract: We have previously demonstrated that cytidine5′‐diphosphocholine (CDP‐choline or citicoline) attenuated arachidonicacid (ArAc) release and provided significant protection for the vulnerablehippocampal CA1 neurons of the cornu ammonis after transientforebrain ischemia of gerbil. ArAc is released by the activation ofphospholipases and the alteration of phosphatidylcholine (PtdCho) synthesis.Released ArAc is metabolized by cyclooxygenases/lipoxygenases to formeicosanoids and reactive oxygen species (ROS). ROS contribute to neurotoxicitythrough generation of lipid peroxides and the cytotoxic byproducts4‐hydroxynonenal and acrolein. ArAc can also stimulate sphingomyelinase toproduce ceramide, a potent pro‐apoptotic agent. In the present study, weexamined the changes and effect of CDP‐choline on ceramide and phospholipidsincluding PtdCho, phosphatidylethanolamine (PtdEtn), phosphatidylinositol(PtdIns), phosphatidylserine (PtdSer), sphingomyelin, and cardiolipin (anexclusive inner mitochondrial membrane lipid essential for electron transport)following ischemia/1‐day reperfusion. Our studies indicated significantdecreases in total PtdCho, PtdIns, PtdSer, sphingomyelin, and cardiolipin andloss of ArAc from PtdEtn in gerbil hippocampus after 10‐min forebrainischemia/1‐day reperfusion. CDP‐choline (500 mg/kg i.p. immediately afterischemia and at 3‐h reperfusion) significantly restored the PtdCho,sphingomyelin, and cardiolipin levels as well as the ArAc content of PtdChoand PtdEtn but did not affect PtdIns and PtdSer. These data suggest multiplebeneficial effects of CDP‐choline: (1) stabilizing the cell membrane byrestoring PtdCho and sphingomyelin (prominent components of outer cellmembrane), (2) attenuating the release of ArAc and limiting its oxidativemetabolism, and (3) restoring cardiolipin levels.

Arachidonic acid and leukotriene C4 : Role in transient cerebral ischemia of gerbils

Accumulation of arachidonic acid (AA) is greatest in brain regions most sensitive to transient ischemia. Free AA released after ischemia is either: 1) reincorporated into the membrane phospholipids, or 2) oxidized during reperfusion by lipoxygenases and cyclooxygenases, producing leukotrienes (LT), prostaglandins, thromboxanes and oxygen radicals. AA, its metabolite LTC4 and lipid peroxides (generated during AA metabolism) have been implicated in the blood-brain barrier (BBB) dysfunction, edema and neuronal death after ischemia/reperfusion. This report describes the time course of AA release, LTC4 accumulation and association with the physiological outcome during transient cerebral ischemia of gerbils. Significant amount of AA was detected immediately after 10 min ischemia (0 min reperfusion) which returned to sham levels within 30 min reperfusion. A later release of AA occurred after 1 d. LTC4 levels were elevated at 0–6 h and 1 d after ischemia. Increased lipid peroxidation due to AA metabolism was observed between 2–6 h. BBB dysfunction occurred at 6 h. Significant edema developed at 1 and 2 d after ischemia and reached maximum at 3 d. Ischemia resulted in ~80% neuronal death in the CA1 hippocampal region. Pretreatment with a 5-lipoxygenase inhibitor, AA861 resulted in significant attenuation of LTC4 levels (Baskaya et al. 1996. J. Neurosurg. 85:112–116) and CA1 neuronal death. Accumulation of AA and LTC4, together with highly reactive oxygen radicals and lipid peroxides, may alter membrane permeability, resulting in BBB dysfunction, edema and ultimately to neuronal death.

CDP-choline: neuroprotection in transient forebrain ischemia of gerbils.

CDP‐choline is a rate‐limiting intermediate in the biosynthesis of phosphatidylcholine (PtdCho), an important component of the neural cell membrane. The ability of CDP‐choline to alter phospholipid metabolism is an important function in the treatment of ischemic injury. Exogenous treatment with CDP‐choline stimulates PtdCho synthesis and prevents release of free fatty acids (FFA), especially arachidonic acid (AA), after ischemia/reperfusion. Phase III clinical trials of CDP‐choline in the treatment of stroke are currently underway. Here we report the neuroprotection by CDP‐choline in transient forebrain ischemia of gerbils. CDP‐choline significantly attenuated the blood‐brain barrier (BBB) dysfunction after ischemia with 6‐hr reperfusion, and considerably reduced the increase of AA in FFA and leukotriene C4 (LTC4) synthesis at 1 day. Edema was significantly elevated after 1 and 2 days, but attained maximum at 3‐day reperfusion. CDP‐choline substantially attenuated edema at 3 days. Ischemia resulted in 80 ± 8% CA1 hippocampal neuronal death after 6‐day reperfusion, and CDP‐choline provided 65 ± 6% neuroprotection. CDP‐choline may act by increasing PtdCho synthesis via two pathways: (1) conversion of 1,2‐diacylglycerol to PtdCho, and (2) biosynthesis of S‐adenosyl‐L‐methionine, thus stabilizing the membrane and reducing AA release and metabolism to leukotriene C4. This would result in decreased toxicity due to AA, leukotrienes, oxygen radicals, lipid peroxidation, and altered glutamate uptake, thus limiting BBB dysfunction, edema and providing neuroprotection. J. Neurosci. Res. 58:697–705, 1999.

Effects of MDL 72527, a specific inhibitor of polyamine oxidase, on brain edema, ischemic injury volume, and tissue polyamine levels in rats after temporary middle cerebral artery occlusion

Abstract The possible effects of the polyamine interconversion pathway on tissue polyamine levels, brain edema formation, and ischemic injury volume were studied by using a selective irreversible inhibitor, MDL 72527, of the interconversion pathway enzyme, polyamine oxidase. In an intraluminal suture occlusion model of middle coerebral artery in spontaneously hypertensive rats, 100 mg/kg MDL 72527 changed the brain edema formation from 85.7 ± 0.3 to 84.5 ± 0.9% in cortex (P < 0.05) and from 79.9 ± 1.7 to 78.4 ± 2.0% in subcortex (difference not significant). Ischemic injury volume was reduced by 22% in the cortex (P < 0.05) and 17% in the subcortex (P < 0.05) after inhibition of polyamine oxidase by MDL 72527. There was an increase in tissue putrescine levels together with a decrease in spermine and spermidine levels at the ischemic site compared with the nonischemic site compared with the nonischemic site after ischemia‐reperfusion injury. The increase in putrescine levels at the ischemic cortical and subcortical region was reduced by a mean of 45% with MDL 72527 treatment. These results suggest that the polyamine interconversion pathway has an important role in the postischemic increase ini putrescine levels and that blocking of this pathway can be neuroprotective against neuronal cell damage after temporary focal cerebral ischemia.

Fluorometric assay of nitrite and nitrate in brain tissue after traumatic brain injury and cerebral ischemia

Nitric oxide synthase (NOS) is distributed within the brain, and nitric oxide (NO) is felt to be involved in the pathophysiology of deterioration after head injury and cerebral ischemia. This study determined the levels of the stable end products of NOS (NOx=nitrite+nitrate) after traumatic brain injury (TBI) and transient cerebral ischemia. A fluorometric assay using nitrate reductase and the NADPH regenerating system was used to quantitate NOx in ultrafiltered (10-kDa cutoff) cortical and hippocampal extracts after reduction of nitrate. In TBI rats, both the plasma and tissue showed a sharp increase in NOx levels 5 min after injury. Plasma NOx returned to control levels by 2 h after injury. Ipsilateral-cortex NOx levels returned to control levels approximately 6 h after injury and remained constant from 6-24 h. Contralateral-cortex returned near to control levels after 1 h. Hippocampus also followed a similar trend. In gerbils, there was a significant elevation in tissue NOx levels immediately after 10 min transient cerebral ischemia, which gradually returned to control levels over 24 h reperfusion. This striking burst of NO synthesis immediately after injury is clearly evident whether the injury is head trauma or ischemia, or whether the measurements were performed on tissue or plasma. It is unknown whether endothelial NOS, neuronal NOS, or both caused the elevation of the NO end products seen after the CNS insults.

Neuroprotection by group I metabotropic glutamate receptor antagonists in forebrain ischemia of gerbil

Stimulation of group I metabotropic glutamate receptors (mGluR 1 and 5) activates G-protein coupled-phospholipase C (PLC) to release 1,2-diacylglycerol (DAG) and arachidonic acid (ArAc). To elucidate the role of group I mGluR, we tested the effects of (S)-alpha-methyl-4-carboxy-phenylglycine (MCPG, mGluR 1 and 5 antagonist), 1-aminoindan-1,5-dicarboxylic acid (AIDA, mGluR 1a specific antagonist) and 2-methyl-6-(phenylethynyl) pyridine (MPEP, mGluR 5 antagonist) on ArAc release and neuronal survival after transient forebrain ischemia in gerbils. Ischemia resulted in (a) significant release of ArAc at 1-day reperfusion and (b) significant neuronal death in the hippocampal CA1 subfield after 6-day reperfusion. MCPG and MPEP decreased ArAc release and also significantly increased neuronal survival. AIDA was less effective in decreasing ArAc release and had no effect on neuronal death. These results suggest that activation of mGluR 5 may be an important pathway in ArAc release and neuronal death after transient ischemia.

Regional activity of ornithine decarboxylase and edema formation after traumatic brain injury.

This study examined ornithine decarboxylase (ODC) activity and edema formation bilaterally in brain cortices and hippocampi after lateral controlled cortical-impact injury in rats. To measure the activity of ODC, the brains of injured and control rats were frozen in situ at 30 minutes and at 6, 24, and 72 hours after controlled cortical-impact injury of moderate severity. The specific gravity of these regions was examined in decapitated animals at corresponding time points as an indicator of edema formation. Thirty minutes after injury, ODC activity did not increase in the injury-site cortex and ipsilateral hippocampus. At 6 hours after injury, ODC activity had increased by nine times that of the control in the injury-site cortex, by five times in the adjacent cortex, and by five and one-half times in the ipsilateral hippocampus. Twenty-four hours after injury, ODC activity had increased by three times that of the control in the injury-site cortex and two times in the ipsilateral hippocampus. Seventy-two hours after injury, activity had returned to control levels. ODC activity increased significantly in the contralateral cortex and hippocampus only at 6 and 24 hours. The injury-site and adjacent cortices and the ipsilateral hippocampus showed significant edema at 6, 24, and 72 hours but not at 30 minutes after injury. These findings indicate that polyamine metabolism is significantly altered in traumatic brain injury. The temporal association between ODC activity and edema formation indicates that polyamines might be a contributing factor in edema formation after traumatic brain injury. The delayed induction of ODC after brain injury suggests a potential therapeutic window for future pharmacological intervention to decrease posttraumatic secondary cerebral injury.

Does CDP-choline modulate phospholipase activities after transient forebrain ischemia?

Ten min forebrain ischemia/1-day reperfusion resulted in significant decreases in total phosphatidylcholine (PtdCho), phosphatidylinositol (PtdIns), and cardiolipin in gerbil hippocampus. CDP-choline restored cardiolipin levels, arachidonic acid content of PtdCho, partially but significantly restored total PtdCho, and had no effect on PtdIns. These data suggest that CDP-choline prevented the activation of phospholipase A(2) (rather than inhibiting phospholipase A(2) activity) but did not affect activities of PtdCho-phospholipases C and/or D, or phosphoinositide-phospholipase C. CDP-choline also provided significant protection for hippocampal CA(1) neurons.

Elevated N1-acetylspermidine levels in gerbil and rat brains after CNS injury

The polyamine system is very sensitive to different pathological states of the brain and is perturbed after CNS injury. The main modifications are significant increases in ornithine decarboxylase activity and an increase in tissue putrescine levels. Previously we have shown that the specific polyamine oxidase (PAO) inhibitor N1,N4‐bis(2,3‐butadienyl)‐1,4‐butanediamine (MDL 72527) reduced the tissue putrescine levels, edema, and infarct volume after transient focal cerebral ischemia in spontaneously hypertensive rats and traumatic brain injury of Sprague‐Dawley rats. In the present study, N1‐acetylspermidine accumulation was greater in injured brain regions compared with sham or contralateral regions following inhibition of PAO by MDL 72527. This indicates spermidine/spermine‐N1‐acetyltransferase (SSAT) activation after CNS injury. The observed increase in N1‐acetylspermidine levels at 1 day after CNS trauma paralleled the decrease in putrescine levels after treatment with MDL 72527. This suggests that the increased putrescine formation at 1 day after CNS injury is mediated by the SSAT/PAO pathway, consistent with increased SSAT mRNA after transient ischemia.