Effect of nitroprusside on regional cerebral cyclic GMP, blood flow and O2 consumption in rat

This investigation was conducted to test whether topical nitroprusside (NP), a cytosolic guanylate cyclase activator, would increase the level of cyclic GMP and alter O₂ consumption or blood flow in the cerebral cortex of rats. Male Long-Evans rats were used in a control (n = 9), low dose NP (n = 13, 10⁻³ M) or high dose NP (n = 12, 10⁻² M) group. Nitroprusside or saline was topically applied to the right side of the cerebral cortex and the left side was used as a control. The cyclic GMP level was determined in five rats in each group using a radioimmunoassay. In the o ther rats in each group, regional cerebral blood flow was measured by [¹⁴C]iodoantipyrine and regional arterial and venous O₂ saturations were determined microspectrophotometrically. Nitroprusside significantly increased the cyclic GMP level from 21.4 ± 12.0 pmol/g (contralateral cortex) to 52.2 ± 36.7 pmol/g (NP treated cortex) in low dose nitroprusside group and from 19.9 ± 22.6 pmol/g (contralateral cortex) to 58.5 ± 15.1 pmol/g (NP treated cortex) in high dose nitroprusside group. High dose nitroprusside significantly increased cerebral blood flow from 80 ± 11 ml · min⁻¹ · 100 g (contralateral cortex) to 114 ± 11 ml · min⁻¹ · 100 g (NP treated cortex). However, there was no significant difference in O₂ extraction and O₂ consumption between the NP treated cortex and contralateral cortex in either the low or the high dose NP groups. In the high dose NP group, the O₂ extraction was 8.0 ± 1.3 ml O₂ · 100 ml⁻¹ in the treated cortex and 8.8 ± 1.5 ml O₂ · 100 ml⁻¹ in the contralateral cortex, while the O₂ consumptions in the NP treated cortex and contralateral cortex were 8.1 ± 1.3 ml O₂ · min⁻¹ · 100 g⁻¹ and 7.3 ± 1.2, respectively. Thus, NP increased the cyclic GMP level without a significant change in O₂ consumption in the cerebral cortex. Our data suggested that O₂ consumption in the cerebral cortex was not affected by the increased level of cyclic GMP.     

Effects of hyperosmolar mannitol on regional oxygen supply and consumption in the newborn pig

Abstract

Previous work indicated that opening the blood-brain barrier with hyperosmotic mannitol decreased local venous O₂ saturation and increased cerebral O₂ consumption. This study was performed to assess the vascular effect of hypertonic mannitol on oxygen supply/consumption balance in the newborn pig and to determine the role of nitric oxide in mediating the effects of mannitol. Animals were anesthetized with (J.chloralose and mechanically ventilated to maintain their blood gases within normal range. Retrograde catheterization of the right carotid artery was performed to inject 12 ml to 25% mannitol over a 30 sec interval. In one group of animals (n = 5), the blood-brain barrier transfer coefficient (Ki) to ¹⁴C-a aminoisobutyric acid or 14C-urea (n = 4) was measured 72 min after mannitol. In another group of animals (n = 9), regional cerebral blood flow and small vein O₂ saturation was measured using 14C-iodoantripyrine and microspectrophotometry. Similar measurements were made in other groups of animals (n = 9) after pretreatment with 10 mg kg^-1 i.v. of N-omega-nitro-L-arginine methyl ester (L-NAME), 20 min before mannitol injection. The mannitol injection did not increase Ki or local cerebral O₂ consumption. It resulted in a decreased small vein O₂ saturation in the ipsilateral cortex (46 ± 3%) in comparison to the contralateral cortex (55 ± 2%). The O₂ supply/consumption ratio decreased in the ipsilateral cortex in the mannitol injected animals (2.14 ± 0.23) in comparison to the contralateral cortex (2.76 ± 0.28). Pretreatment with L-NAME abolished this effect of mannitol (small vein O₂ saturation 59 ± 2% in ipsilateral cortex and 58 ± 2% in the contralateral cortex; O₂ supply/consumption 2.68 ± 0.17 in the ipsilateral cortex and 2.65 ± 0.16 in the contralateral cortex). We conclude that hypertonic mannitol adversely affects O₂ supply/consumption balance, without increasing bloQd-brain barrier transport, and this effect is blocked by L-NAME, a nitric oxide synthase antagonist. [Neural Res 1997; 19: 204–210]     

Influence of lesions of the noradrenergic locus coeruleus system on the cerebral metabolic response to bicuculline-induced seizures

The objective of the present study was to explore if lesions of the ascending noradrenergic pathways, originating in the locus coeruleus, modulate the cerebral metabolic response to bicuculline-induced seizures in rats. Bilateral noradrenergic lesions were performed by 6-hydroxydopamine injections in the caudal mesencephalon, 12–22 days before seizures were induced in animals ventilated on N₂O:O₂ (75:25). After 5 min of seizures the brain was frozen in situ and cerebral cortex and hippocampus were sampled for analysis. Labile phosphates, glycolytic metabolites, cyclic nucleotides, and free fatty acids were measured. In another series, lesioned animals were used for measurements of cerebral oxygen consumption.The noradrenergic lesions neither modified the electroencephalographically recorded seizure discharge, nor did they alter cerebral oxygen consumption or cerebral energy state. However, when compared to sham-operated animals, those with noradrenergic lesions had significantly higher (115% and 68%) glycogen concentrations and lower (50% and 52%) cyclic AMP concentrations in cerebral cortex and hippocampus, respectively, demonstrating the marked influence of noradrenergic activity on adenylate cyclase activity and glycogenolysis. The lesions failed to modulate the rise in free fatty acids in the cerebral cortex, or the cyclic GMP concentrations in the cerebral cortex and hippocampus. Thus, increased noradrenergic activity during status epilepticus does not seem responsible for lipolysis or for activation of guanylate cyclase.     

17Beta-estradiol suppresses AMPA-induced increases in regional cerebral O2 consumption

Abstract

We tested the hypothesis that 17beta-estradiol would reduce the cerebral O₂ consumption response resulting from glutamate receptor stimulation by alpha amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA). Fourteen ovariectomized rats were separated into 17beta-estradiol (0.5 mg 21 day release pellet) and control (placebo pellet) groups to determine cerebral blood flow (¹⁴C-iodoantipyrine) and O₂ consumption (microspectrophotometry). After topical cortical stimulation with 10^-3 M and 10-4 M AMPA, cerebral blood flow increased significantly in both groups in a concentration-dependent manner. Cerebral O₂ extraction was not significantly different in any region of the 17beta-estradiol treated group. In the placebo treated group, the O₂ extraction in the saline treated cortex and in the 10^-3 M AMPA treated cortex was significanly higher when compared to the 10^-4 M AMPA treated cortex. Cerebral O₂ consumption in the control group increased by 20%, from 5.2 ± 0.6 to 6.1 ± 0.7, with 10^-4 M AMPA and significantly increased by 64% to 8.5 ± 0.8 ml O₂ min^-1 100 g^-1 with 10^-3 M AMPA. The 17beta-estradiol group demonstrated no statistically significant difference in O₂ consumption between the saline treated and AMPA treated cortex. Thus, 17beta-estradiol reduced the effects of AMPA in increasing cerebral O₂ consumption.     

Improvement in Microregional Oxygen Supply/Consumption Balance and Infarct Size After Cerebral Ischemia-Reperfusion With Inhibition of p70 Ribosomal S6 Kinase (S6K1)

Background: We tested the hypothesis that inhibition of p70 ribosomal S6 kinase (S6K1) would decrease infarct size and improve microregional O₂ supply/consumption balance after cerebral ischemia-reperfusion. Methods: This was tested in isoflurane-anesthetized rats with middle cerebral artery blockade for 1 hour and reperfusion for 2 hours with or without PF-4708671 (S6K1 inhibitor, 75 mg/kg, 15 minutes after blockade). Regional cerebral blood flow was determined using a C¹⁴-iodoantipyrine autoradiographic technique. Regional small vessel (20-60 μm diameter) arterial and venous oxygen saturations were determined microspectrophotometrically. Results: There were no significant hemodynamic or arterial blood gas differences between groups. The control ischemic-reperfused cortex had a similar O₂ consumption to the contralateral cortex. However, microregional O₂ supply/consumption balance was significantly reduced in the ischemic-reperfused cortex with many areas of low O₂ saturation (23 of 80 veins with O₂ saturation below 45%). PF-4708671 did not significantly alter cerebral blood flow or O₂ consumption. However, it significantly reduced the number of small veins with low O₂ saturations in the reperfused region (6 of 80 veins with O₂ saturation below 45%). This was associated with a significantly reduced cortical infarct size after S6K1 inhibition (12.9 ± .8% control versus 6.6 ± .3% PF-4708671). Conclusion: This suggests that S6K1 inhibition is important for cell survival and that it reduces the number of small microregions with reduced local oxygen balance after cerebral ischemia-reperfusion.     

颅骨修补前后脑血流变化的CT灌注评价

目的探讨颅骨修补手术前后患者脑血流的变化。方法对24例早期行颅骨修补手术患者,在颅骨修补前1～2d和术后10～14d,应用16排螺旋CT做脑灌注扫描,记录大脑皮层、基底节和丘脑区域灌注图像上rCBV、rCBF、MTT及TTP等参数值。分析颅骨修补前后双侧大脑皮质,基底节和丘脑相关区域血流灌注数据的变化。结果颅骨修补术前患侧皮质区CBF明显低于对侧,和健侧相比差异具有统计学意义(P<0.01);术后患侧在皮质区CBF显著上升,和术前相比,差异具有统计学意义(P<0.01)。两侧基底节和丘脑区在术前术后脑血流相关数值虽有所上升,但前后相比差异无统计学意义(P>0.05)。结论头颅CT灌注可以及时检测颅脑外伤后不同阶段脑组织血流量的变化,而颅骨修补术可以明显提高患侧大脑皮质区的脑血流量,进而可能促进神经功能恢复。     

A nonNMDA antagonist,GYKI 52466 improves microscopic O2 balance in the cortex during focal cerebral ischemia

Abstract

This study was performed to test whether GYKI 52466, a nonNMDA receptor antagonist; would improve microregional oxygen supply and consumption balance in the focal cerebral ischemic area. Rats were anesthetized with 1.4% isoflurane. For the GYKI Group (n = 8), 10 min before middle cerebral artery (MCA) occlusion, a bolus of 5 mg kg^–1 of GYKI 52466 i.v. was administered and was followed by an infusion of 5 mg kg^–1 h^–1. For the Control Group (n = 8), the same volume of the vehicle was administered. One hour after MCA occlusion, regional cerebral blood flow (rCBF) was measured using the 14C-iodoantipyrine autoradiographic technique. Microscopic arterial and venous oxygen saturations were determined using microspectrophotometry. In the cortex contralateral to MCA occlusion, the average rCBF and the average 02 consumption were lower in the GYKI Group than in the Control Group (rCBF: GYKI 65.5 dh 24.1 ml 100 g^–1 min^–1 Control 97.7±33 A ml 100g^–1 min^–1; 02 consumption: GYKI 3.9 ± 1.2 ml 02 100 g 1 min^–1 Control 6.2 ±2.5 ml 02 100g 1 min^–1) without a significant difference in the number of veins with Sv02 < 50%. In the ischemic cortex, the number of veins with Sv02 < 50% was significantly smaller in the GYKI Group (21 veins out of 63) than in the Control Group (45 out of 59) without a significant difference in the average rCBF (GYKI 44.9±17.7, Control 29.7±10.4) or regional 02 consumption between these two groups (GYKI 3.3 ± 1.4, Control 2.7± 1.2). Our data demonstrated that GYKI 52466 was effective in improving microscopic 02 balance in the focal ischemic cortical area of the brain and it decreased 02 consumption in the non-ischemic cortex. [Neurol Res 1999; 21: 299-304]     

Effect of methylene blue on middle cerebral artery flow velocity in a patient with severe sepsis following clipping of a cerebral aneurysm

Introduction: The use of methylene blue, an inhibitor of guanylate cyclase, has been described in patients with septic shock who are unresponsive to inotropic agents. However, the effects of methylene blue on the human cerebral circulation are not known. Methods and Results: This article presents a case report of a 58-year old-female with a clinical presentation compatible with severe sepsis and increasing inotropic requirements following clipping of a cerebral aneurysm. Administration of methylene blue (2 milligrams/kilograms) intravenously was undertaken with monitoring of mean arterial pressure and middle cerebral artery flow velocity (FVm). The effect of methylene blue on mean arterial pressure occurred quite rapidly after initiation of the infusion, allowing downward titration of norepinephrine. Initially, FVm increased in association with an increase in mean arterial pressure, reaching its highest value halfway through the infusion. Subsequently, FVm decreased to baseline by the end of the monitoring period. The rise in mean arterial pressure and increase in FVm were accompanied by a reduction in cerebral vascular resistance assuming intracranial pressure, and the diameter of the insonated vessel was unchanged during and immediately after infusion of methylene blue. Conclusion: Methylene blue did not appear to have a major untoward effect on cerebrovascular resistance in this patient. The limited characterization of cerebrovascular effects provided by this article mandates the need for careful monitoring of cerebrovascular behavior and adequacy during use of methylene blue.     

Effect of up-regulation of NMDA receptors on cerebral O2 consumption and blood flow in rat

We tested the hypothesis that cerebrocortical blood flow and O₂ consumption would be proportional to an up-regulated number of functional N-methyl- -aspartate (NMDA) receptors. Previous work had shown a relationship between cerebral metabolism and NMDA receptor activity. We increased the specific binding to NMDA receptors in the cerebral cortex, from 2.2 ± 0.9 to 4.5 ± 0.8 (density units) in male Long-Evans rats by daily giving two intraperitoneal injections (30 mg/kg) of CGS-19755, an NMDA receptor inhibitor, for 7 consecutive days (discontinued for 20 h before experiment). Twelve up-regulated (CGS treated) and 12 control rats were used in this study. Under isoflurane anesthesia and after topical stimulation of the right cerebral cortex with 10⁻² M NMDA, the blood flow (¹⁴C-iodoantipyrine method) increased from 98 ± 11 ml/min/100 g in the unstimulated cortex of the control rats to 161 ± 37 ml/min/100 g in the stimulated cortex. The unstimulated value for blood flow (95 ± 7 ml/min/100 g) did not change in the upregulated group but it doubled (194 ± 69 ml/min/100 g) in the stimulated, upregulated cortex. Similarly, O₂ consumption (cryomicrospectrophotometrically determined) in normal rats increased 46%, from 9.3 ± 1 ml/min/100 g to 13.6 ± 4 after NMDA stimulation. While in the upregulated animals, O₂ consumption increased 103% from 7.9 ± 0.6 to 16 ± 6.5 after NMDA stimulation. In conclusion, NMDA receptor upregulation does not alter basal cerebrocortical blood flow or O₂ consumption but in the NMDA-stimulated cortex, the blood flow and O₂ consumption increase is dependent on the number of NMDA receptors present.     

The role of language areas in motor control dysfunction in Parkinson's disease

We evaluated the differences in motor control organization between parkinsonian patients with (seven cases) and without (ten cases) gait disorder. We used positron emission tomography (O¹⁵-H₂O-PET) to measure regional cerebral blood flow as a correlate for local neuronal activation. This has been assessed during repetitive joystick movements of the right hand, externally triggered using a metronome as an auditory cue. In patients with Parkinson's disease (PD) without gait disorder, the contralateral supplementary motor cortex and the bilateral cerebellum were activated while in PD patients with gait disorder the contralateral Broca's area, the contralateral sensory motor cortex and the homolateral cerebellum were activated. Our results suggest that PD patients with gait disorder creates an internal verbal cue in order to control the output of the movement of joystick, supplying the loss of control of the supplementary motor cortex that is activated in patients without gait disorder.     

Mathematical model of oxygen transport in the cerebral cortex

To estimate the magnitude of hyperemia necessary to support oxidative metabolism in the cerebral cortex during functional activation, a mathematical model of O₂ transport from capillary to tissue was developed. Radial and axial gradients of O₂ pressure in tissue surrounding a single capillary were calculated at normal and increased cerebral metabolic rates for O₂. Cone-shaped tissue geometry and nonlinear oxyhemoglobin dissociation were assumed. Local O₂ consumption was assumed to be supported with local tissue pO₂ greater than 1 mmHg. The distribution of tissue pO₂ was also calculated during moderate hypoxemia (p_aO₂=42 mmHg), using experimental values of red blood cell velocity measured in individual capillaries of the rat cerebral cortex using intravital video-microscopy. The model predicted that moderate increases (≤50%) in cerebral O₂ consumption were supported by proportional increases in capillary blood flow. Large increases in O₂ consumption (50–110%) were supported by disproportional increases in flow. During moderate hypoxemia, average tissue pO₂ decreased but oxygen utilization was sustained when capillary flow was increased to a level measured in experiments. The results suggest a proportional relationship between cerebrocortical blood flow and oxygen consumption in the normal physiological range of functional activation.     

Observations on cerebral ischaemia in cats at injury threshold levels

Abstract

The potential for recovery of brain tissue subjected to ischaemia at a threshold level of injury was evaluated in cats subjected to 20 min middle cerebral artery occlusion. In addition to assessment of regional cerebral blood flow and water content, the permeability of the bloodbrain barrier and morphological changes detected by light microscopy were studied at various time intervals. Our observations revealed that although a similar reduction of blood flow during arterial occlusion was found both in the caudate nucleus and the cerebral cortex, the reactive hyperaemia was consistently higher in the caudate nucleus than in the cortex. After 24 h the caudate nucleus also revealed a significantly higher water content and increased vascular permeability than the cortex. Morphological observations at 24 h in areas affected by ischaemia showed widespread, marked ischaemic neuronal injury, whereas at 3 d there was, in addition, a vigorous proliferative reaction of vascular elements. Cats sacrificed at 14 d revealed a remarkably good preservation of neurons, both in the caudate nucleus and cortex which otherwise showed a few circumscribedsmall, infarcts surrounded by normal nerve cells. Our study suggests that neurons injured at threshold level have a considerable capacity for recovery. Otherwise, with a similar degree of ischaemiathe caudate nucleus appears more prone to increased vascular permeability and oedema than the cerebral cortex.     

Comparison of Glucose Metabolism and Cerebral Blood Flow During Cortical Motor Activation

Regions of cerebral cortex activated in normal subjects making simple, repetitive, voluntary wrist movements were studied with positron emission tomography (PET). The regional cerebral metabolic rate of glucose utilization was studied with 2-[¹⁸F]fluoro-2-deoxy-D-glucose (FDG), and regional cerebral blood flow was studied with ¹⁵O-labeled water. No significant activation was found with the cerebral metabolic rate studies. Studies of regional cerebral blood flow showed significant activation of the contralateral sensorimotor cortex region of 42%, of the ipsilateral sensorimotor cortex region of 19%, and of the medial frontal cortex of 30% compared with the resting state. Increases in blood flow in the contralateral sensorimotor cortex and medial frontal cortex were visible on every activated scan. Measurement of regional cerebral blood flow seems to be more sensitive than regional cerebral metabolic rate of glucose utilization for studying cortical activation with voluntary movement.     

In vivo disturbance of the oxidative metabolism of glucose in human cerebral gliomas

Abnormalities in the oxidative metabolism of glucose in human cerebral gliomas have been studied in seven patients using positron emission tomography. Measurements of regional cerebral blood flow and oxygen consumption were obtained using the oxygen-15 steady-state inhalation technique. Values of regional cerebral glucose consumption were obtained using fluorine 18-labeled 2-fluoro-2-deoxy-D-glucose and a simplification of the method of Sokoloff. Functional values were obtained for regions of tumor and brain tissue in the middle cerebral artery territory of the contralateral cortex. Values of regional glucose consumption were calculated for both regions using a value of the lumped constant quoted for normal brain tissue (0.42). Tumor regional cerebral blood flow was comparable to that in the contralateral cortex, whereas regional cerebral oxygen consumption was depressed. This depression resulted in low tumor values of the fractional oxygen extraction ratio (0.21 +/- 0.07), indicating that oxygen supply exceeded the metabolic demand. In contrast, tumor regional cerebral glucose consumption was not depressed and regional glucose extraction ratios were similar for tumor and brain tissue. The metabolic uncoupling between regional oxygen consumption and regional glucose consumption (CMRO2/CMRGlu = 0.24 +/- 0.07 ml of oxygen per milligram of glucose) is indicative of increased aerobic glycolysis.     

Increased cerebral oxygen consumption in Eker rats and effects of N-methyl-D-aspartate blockade: Implications for autism

Because there is a strong correlation between tuberous sclerosis and autism, we used a tuberous sclerosis model (Eker rat) to test the hypothesis that these animals would have an altered regional cerebral O2 consumption that might be associated with autism. We also examined whether the altered cerebral O2 consumption was related to changes in the importance of N-methyl-D-aspartate (NMDA) receptors. Young (4 weeks) male control Long Evans (N = 14) and Eker (N = 14) rats (70-100 g) were divided into control and CGS-19755 (10 mg/kg, competitive NMDA antagonist)-treated animals. Cerebral regional blood flow (14C-iodoantipyrine) and O2 consumption (cryomicrospectrophotometry) were determined in isoflurane-anesthetized rats. NMDA receptor protein levels were determined by Western immunoblotting. We found significantly increased basal O2 consumption in the cortex (6.2 +/- 0.6 ml O2/min/100 g Eker vs. 4.7 +/- 0.4 Long Evans), hippocampus, cerebellum, and pons. Regional cerebral blood flow was also elevated in Eker rats at baseline, but cerebral O2 extraction was similar. CGS-19755 significantly lowered O2 consumption in the cortex (2.8 +/- 0.3), hippocampus, and pons of the Long Evans rats but had no effect on cortex (5.8 +/- 0.8) or other regions of the Eker rats. Cerebral blood flow followed a similar pattern. NMDA receptor protein levels (NR1 subunit) were similar between groups. In conclusion, Eker rats had significantly elevated cerebral O2 consumption and blood flow, but this was not related to NMDA receptor activation. In fact, the importance of NMDA receptors in the control of basal cerebral O2 consumption was reduced. This might have important implications in the treatment of autism.     

Cerebrovascular response to intracarotid injection of serotonin before and after middle cerebral artery occlusion

The effect of intracarotid injection of serotonin (5-HT) on internal carotid artery flow and oxygen availability (O₂a) of the cerebral cortex was studied in 10 baboons. Vasoconstriction occurred in the vascular bed of the territory supplied by the injected artery. After one middle cerebral artery was occluded the vasoconstrictor effect of 5-HT was more pronounced, particularly in the non-ischaemic hemisphere. The capacity of the cerebral vessels to provide collateral blood flow was reduced in both ischaemic and non-ischaemic areas of brain. As a result of focal cerebral ischaemia, 5-HT may accumulate in the brain and contribute to the progression of infarction.     

Redistribution of blood flow in the cerebral cortex of normal subjects during head-up postural change

Regional cerebral blood flow was measured in 21 normotensive subjects during supine rest and during head-up tilt to 70°. The results showed significant and consistent regional cerebral blood flow changes in the frontal areas with lower relative flow distribution values (percentage of mean flow) during head-up tilt than during supine rest. The lower frontal flow distribution values during tilt were not related to habituation, to repeated measurements, or to the estimated level of arterial CO₂ which was derived from expired end-tidal CO₂ levels. None of the subjects had orthostatic hypotension and there was no significant difference in mean hemispheric blood flow between lying down and standing up. There was no significant gender difference in regional cerebral blood flow, although female subjects tended to have higher mean hemispheric flow than males in both postures. It remains to be established whether the flow decreases in the frontal cortex are caused by cerebral functional factors or by haemodynamic mechanisms.     

Stability of Oxygen Transmissibility During Ischemia

Abstract

Simultaneous measurements of oxygen availability (O₂a) and partial pressure (PO₂) were made by the double noble metal electrode system designed by Erdmann and Krell in an attempt to monitor the oxygen transmissibility (Dk) with respect to time before and after ischemic episodes in the brain cortex. Available current equations can appropriately describe the performance of the electrode in a homogeneous nonconsuming medium, and with some experimental error this electrode system can be used to estimate oxygen transmissibility changes in the brain based on the different chararcteristics of the O₂a (rod-type, flush-ended bare tip) and the PO₂ (rod-type, recessed tip) cathodes. Our method monitors the oxygen transmissibility within the diffusion sphere of the bare tip as the ratio of the currents related to O₂a and PO₂. We find with this system that even prolonged global cerebral ischemia does not alter the oxygen transmissibility of the brain tissue. Therefore, alteration of the oxygen transmissibility can not be a contributing factor to postischemic cerebral malfunction.     

Guanylate cyclase—Cyclic GMP in mouse cerebral cortex and cerebellum: Modification by anticonvulsants

Incubated tissue slices from mouse cerebral cortex and cerebellum readily accumulate cyclic GMP in response to a challenge by ouabain, NaN₃, NH₂OH, or KCl. Under similar conditions, l-glutamate, l-aspartate, glycine, γ-aminobutyric acid, kainic acid, and the calcium ionophore, A-23187, were ineffective. Inhibition of the ouabain-induced accumulation of cyclic GMP was evident with valproate, carbamazepine, clonazepam, phenytoin, and phenobarbital. Only phenytoin blocked the action of KCl and cyclic GMP responses to NaN₃ were inhibited by high concentrations of valproate, carbamazepine, phenytoin, or phenobarbital. The effects of NH₂OH were attenuated by high concentrations of carbamazepine, phenobarbital, and clonazepam (cortex only). Guanylate cyclase activity in homogenates of cortex and cerebellum was enhanced in the presence of NaN₃, NH₂OH, or Ca²⁺ (in low concentrations of Mn²⁺). The enzyme activation induced by Ca⁺ was blocked only by large (1 mm) amounts of carbamazepine. In like manner, large concentrations of carbamazepine, phenytoin (cortex), or clonazepam (cortex) were effective in reducing guanylate cyclase stimulation by NaN₃. No agent affected the NH₂OH responses. The results suggest that anticonvulsant drug actions with regard to central cyclic GMP systems are related to the Na⁺-induced depolarization of nerve tissue and not to any direct actions on the guanylate cyclase enzyme.     

Distinct proteins in cortex of rats with closed traumatic brain injury detected by a WCX-2 protein chip

BACKGROUND: Mechanical injury can cause the changes of polygene expression spectrum in rat cerebral cortical nerve cells, and then result in the changes of intracellular protein expression. At present, dielectrophoresis is combined with mass spectrum technique to detect the expression of different proteins in rat cortex after brain injury, but the protein chip technique requires further investigation. OBJECTIVE: To analyze the differences of protein expression spectrum in rat cerebral cortex before and after closed traumatic brain injury using WCX-2 protein chip technique. DESIGN: A randomized controlled animal experiment. SETTING: Training Division of the Medical College of Chinese People's Armed Police Force. MATERIALS: Seventy-two male SD rats of clean degree, 350–450 g, were provided by the Experimental Animal Center, Academy of Military Medical Sciences of Chinese PLA. Urea, trifluoroacetic acid, CHAPS and Tris (Sigma, USA); WCX-2 (Ciphergen, USA). Ultra-high speed hypothermia centrifuger (Bechman, USA); Rotary tissue microtome (Keuca, Germany); Biochip processor and PBSⅡ-C protein chip reader (Ciphergen, USA). METHODS: The experiments were carried out in the Institute of Molecular Pathology, Central Laboratory, and Department of Pathology, Medical College of Chinese People's Armed Police Force from June 2005 to March 2006. ① Grouping and treatment: The experiments were completed in molecular pathological institute, central laboratory and pathological department. ① The rats were randomly divided into control group (n =12) and brain injury group (n =60). Marmarou's weight-dropping models were duplicated at different time points in the brain injury group. In the control group, the rats were only treated by incising the skin of head top, without fixing the stainless steel hitting backup plate at the vault of skull, and obtain brain cortex for pathological and protein chip research, and they were killed after 24 hours. The rats in the brain injury group were killed at 4, 8, 12, 24 and 48 hours after model establishment. ② Pathological observation: Longitudinal section was made on cerebral cortex, and sections of 5 μm were prepared, then stained with hematoxylin and eosin (HE). ③ Protein chip analysis: 100 mg cerebral cortex was collected from each rat, and the protein content in sample was detected with Bradford method, meanwhile, WCX-2 protein chip was used to analyze the protein spectrum. The data were automatically collected with Ciphergen proteinchip 3.0 software, and the results were analyzed using Biomarker Wizard software to compare the differences of protein spectrum in rat cortex between the groups. MAIN OUTCOME MEASURES: Results of the pathological observation of cerebral cortex and the protein spectrum analysis. RESULTS: ① Pathological changes of cerebral cortex: In the control group, no necrosis and edema was observed. In the brain injury group, injures of different severity occurred at different time points; After 4 hours, focal or scattered red nerve cells could be observed, the size of some cells was increased, cytoplasm was lightly stained, and only nuclear fragments were seen; After 8 hours, the necrotic nerve cells were increased, and the number of nerve cells was reduced, astrocytes (neuronophagia) could be seen in partial cytoplasm; there was small vascular dilatation, and endothelial cell proliferation; interstitial edema, regional rarefaction lightly stained. After 12–48 hours, the necrotic nerve cells were reduced, and astrocytes proliferated. ② Results of protein spectrum analysis: The WCX-2 experiment found that the expressions of 5 639, 3 212 and 7 536 u proteins in cerebral cortex changed after injury in the brain injury group. The peak intensity of 5 639 u protein in the brain injury group at 8 hours after injury was higher than that in the control group (P < 0.05); The peak intensity of 3 212 u protein in the brain injury group at 48 hours after injury was higher than that in the control group (P < 0.05); The peak intensity of 7 536 u protein at 24 hours after injury was higher than that in the control group (P < 0.05). CONCLUSION: Brain injury can cause the changes of protein expression spectrum in cerebral cortex, it is suggested that brain injury can induce the expression of protein.