Zihong Huang

Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2

Phospholipase A2 (PLA2) enzymes are critical regulators of prostaglandin and leukotriene synthesis and can directly modify the composition of cellular membranes,. PLA2 enzymes release fatty acids and lysophospholipids, including the precursor of platelet-activating factor, PAF, from phospholipids. Free fatty acids, eicosanoids, lysophospholipids and PAF are potent regulators of inflammation,,, reproduction and neurotoxicity,,. The physiological roles of the various forms of PLA2 are not well defined. The cytosolic form, cPLA2, preferentially releases arachidonic acid from phospholipids and is regulated by changes in intracellular calcium concentration,. We have now created ‘knockout’ (cPLA2−/−) mice that lack this enzyme, in order to evaluate its physiological importance. We find that cPLA2−/− mice develop normally, but that the females produce only small litters in which the pups are usually dead. Stimulated peritoneal macrophages from cPLA2−/− animals did not produce prostaglandin E2 or leukotriene B4 or C4. After transient middle cerebral artery occlusion, cPLA2−/− mice had smaller infarcts and developed less brain oedema and fewer neurological deficits. Thus cPLA2 is important for macrophage production of inflammatory mediators, fertility, and in the pathophysiology of neuronal death after transient focal cerebral ischaemia.

L-arginine infusion promotes nitric oxide-dependent vasodilation, increases regional cerebral blood flow, and reduces infarction volume in the rat.

Background and Purpose We previously reported that ar-ginine infusion increased pial vessel diameter by nitric oxide-dependent mechanisms, improved regional cerebral blood flow (rCBF) distal to middle cerebral artery (MCA) occlusion, reduced infarction volume in spontaneously hypertensive when administered intraperitoneally before and after MCA occlusion. In this report we extend our findings (1) by exam-ining the time course of L-arginine on rCBF and pial vessel diameter under basal conditions and on rCBF after MCA occlusion and (2) by reproducing the protective effect L-arginine on infarct volume when given intravenously imme-diately after the onset of MCA occlusion in both normotensive and hypertensive models of focal cerebral ischemia. Methods Changes in pial vessel diameter (closed cranial window) and rCBF (laser-Doppler flowmetry) were measured over time after L-arginine infusion into anesthetized Sprague-Dawley rats. rCBF was also measured distal to MCA occlusion in a brain region showing rCBF reductions in the range of 80% of baseline. The effects of infusing L-arginine (300 mg/kg for 10 minutes beginning 5 minutes after occlu-sion) were assessed on infarction volume in Sprague-Dawley rats after proximal MCA occlusion and in spontaneously hypertensive rats after common carotid artery plus distal MCA occlusion.. Results Results L-Arginine (300 mg/kg IV) elevated rCBF by 20% when measured in the dorsolateral cortex of Sprague-Dawley rats and caused L-nitroarginine-methyl ester-inhibitable in-creases in pial vessel diameter. L-Arginine (& 30 mg/kg IV) increased blood flow distal to MCA occlusion by 50%. These effects were sustained throughout the observation period (70 to 105 minutes). Changes in mean arterial blood pressure were not observed. L-Arginine (300 mg/kg IV) reduced infarction volume by 35% and 28% in Sprague-Dawley and spontane-ously hypertensive rats, respectively, when examined 24 hours after vessel occlusion. Conclusions Conclusions These studies extend our previous findings by demonstrating that exogenous L-arginine induces sustained rCBF increases in normal brain as well as in a marginally perfused brain region distal to MCA occlusion. Our data in Sprague-Dawley rats support the conclusion that L-arginine-induced increases in rCBF can decrease infarction volume. We conclude that nitric oxide-mediated mechanisms increase rCBF and decrease infarction volume after MCA occlusion in both normotensive and hypertensive animals. (Stroke. 1994;25:429-435.)

Cerebrovascular responses under controlled and monitored physiological conditions in the anesthetized mouse

Control of physiological parameters such as respiration, blood pressure, and arterial blood gases has been difficult in the mouse due to the lack of technology required to monitor these parameters in small animals. Here we report that anesthetized and artificially ventilated mice can be maintained under physiological control for several hours with apparently normal cerebrovascular reactivity to hypercapnia and mechanical vibrissal stimulation. SV-129 mice were anesthetized with urethane (750 mg/kg i.p.) and α-chloralose (50 mg/kg i.p.), intubated, paralyzed, and artificially ventilated. Respiratory control was maintained within physiological range by reducing the inspiratory phase of the respiratory cycle to <0.1 s and by adjusting end-tidal CO2 to give a Pco2 of 35 ± 3 mm Hg. In these mice, mean arterial pressure (95 ± 9 mm Hg), heart rate (545 ± 78 beats/min), and arterial pH (7.27 ± 0.10) could be maintained for several hours. Body temperature was kept at 36.5–37.5°C. We observed stable regional CBF (rCBF) measurements (as determined by laser–Doppler flowmetry) when systemic arterial blood pressure was varied between 40 and 130 mm Hg. Hypercapnia led to a 38 ± 15% (5% CO2) and 77 ± 34% (10% CO2) increase in rCBF. Mechanical stimulation of contralateral vibrissae for 1 min increased rCBF by 14 ± 4%. Changes in rCBF compare favorably with those observed previously in another rodent species, the Sprague–Dawley rat. After placement of a closed cranial window, cerebrovascular reactivity to hypercapnia and whisker stimulation was intact and well maintained during 2-h superfusion with artificial CSF.

Mismatch between cerebral blood volume and flow index during transient focal ischemia studied with MRI and GD-BOPTA.

We investigated the regional and temporal changes in cerebral blood volume (CBV), cerebral blood flow (CBF), and vascular transit time in seven mongrel cats during 30 min transient focal ischemia, caused by occlusion of the middle cerebral artery. Dynamic susceptibility contrast magnetic resonance imaging was done at 4.7 T, using fast gradient echo T2* weighted imaging and intravenous injection of gadolinium-BOPTA/Dimeglumine. During occlusion, the areas showing a blood volume change were predominantly within the middle cerebral artery territory and could be divided into areas showing either CBV increases or decreases. The area with decreased blood volume also had decreased blood flow as measured by our flow-based index (p < 0.05) and was located in the central territory of the middle cerebral artery. Peripheral to this region was an area showing increased blood volume but without significant CBF changes (p > 0.05). During reperfusion, the CBF increased in the entire zone showing changes in blood volume during occlusion, and remained significantly elevated until 45 min post-occlusion, while CBV remained elevated in the hyperemic rim for at least 2 h. The presence of a peri-ischemic zone showing flow/volume mismatch identified a region wherein baseline CBF is maintained by means of compensatory vasodilatation, but where the ratio of CBF to CBV is decreased. Dynamic susceptibility contrast magnetic resonance imaging with gadolinium-BOPTA/Dimeglumine may be a valuable technique for the investigation of regional and temporal perturbations of hemodynamics during ischemia and reperfusion.

The protein tyrosine phosphatase SHP-2 is expressed in glial and neuronal progenitor cells, postmitotic neurons and reactive astrocytes

In this study we examined the distribution and developmental profile of the src homology 2 (SH2) domain-containing protein tyrosine phosphatase SHP-2 in the mouse brain. We found that SHP-2 is present in both mitotically active and postmitotic cells in the forebrains of embryonic day 12 (E12) mice. In a developmental study extending from embryonic day 12 to adulthood, Western blotting analysis demonstrated equivalent levels of SHP-2 protein at all of the ages examined. Expression of SHP-2 paralleled the level of enzymatic activity at the different developmental periods. In the adult brain SHP-2 was restricted to diverse classes of neurons, while the majority of glial cells did not express detectable levels of protein. However, reactive astrocytes in response to an ischemic brain injury showed SHP-2 immunolabelling. Our data suggest that SHP-2 may play a role in pathways of neuronal and glial progenitor cells, in a broad spectrum of neuronal responses in the adult brain and in the gliotic response to the injury.

Expression of the Transcriptional Repressor Protein Kid-1 Leads to the Disintegration of the Nucleolus

The rat Kid-1 gene codes for a 66-kDa protein with KRAB domains at the NH2 terminus and two Cys2His2-zinc finger clusters of four and nine zinc fingers at the COOH terminus. It was the first KRAB-zinc finger protein for which a transcriptional repressor activity was demonstrated. Subsequently, the KRAB-A domain was identified as a widespread transcriptional repressor motif. We now present a biochemical and functional analysis of the Kid-1 protein in transfected cells. The full-length Kid-1 protein is targeted to the nucleolus and adheres tightly to as yet undefined nucleolar structures, leading eventually to the disintegration of the nucleolus. The tight adherence and nucleolar distribution can be attributed to the larger zinc finger cluster, whereas the KRAB-A domain is responsible for the nucleolar fragmentation. Upon disintegration of the nucleolus, the nucleolar transcription factor upstream binding factor disappears from the nucleolar fragments. In the absence of Kid-1, the KRIP-1 protein, which represents the natural interacting partner of zinc finger proteins with a KRAB-A domain, is homogeneously distributed in the nucleus, whereas coexpression of Kid-1 leads to a shift of KRIP-1 into the nucleolus. Nucleolar run-ons demonstrate that rDNA transcription is shut off in the nucleolar fragments. Our data demonstrate the functional diversity of the KRAB and zinc finger domains of Kid-1 and provide new functional insights into the regulation of the nucleolar structure.