Yong Y. He

Formalin fixation alters water diffusion coefficient magnitude but not anisotropy in infarcted brain

This study was designed to determine whether formalin fixation alters diffusion parameters in the infarcted brain. Diffusion tensor images were obtained from anesthetized mice 1 hr after middle cerebral artery occlusion and repeated after formalin fixation of brains. In live animals, there was a significant decrease in the trace of the diffusion tensor (Tr(D)) in infarcted cortex and external capsule compared with contralateral brain areas, with no change in relative anisotropy (RA). After formalin fixation, Tr(D) was reduced 30–80%. However, the Tr(D) differential present in vivo between injured and healthy tissues was lost, with Tr(D) reduced to similar values in all tissues except for the edge of the cortical infarction, where it was lower than in surrounding tissues. RA values were unchanged after fixation. This study supports the preservation of diffusion anisotropy for both healthy and injured white matter in fixed mouse brain. However, the sensitivity of water diffusion in detecting tissue injury in vivo is not preserved in fixed tissues. Magn Reson Med 53:1447–1451, 2005.

Automated Measurement of Infarct Size With Scanned Images of Triphenyltetrazolium Chloride–Stained Rat Brains

BACKGROUND AND PURPOSE The extent of brain infarction after local cerebral ischemia is frequently assessed with the mitochondrial activity indicator 2,3,5-triphenyltetrazolium chloride (TTC). We describe an automated procedure for analysis of infarct size in TTC-stained rat brains. METHODS Rats were subjected to middle cerebral artery occlusion and killed after 24 to 36 hours, and their brains were processed for TTC staining. Digital images of coronal sections from these brains (n > 50) were acquired with a desktop color scanner. The resulting images were divided into red, blue, and green component images. Total brain and infarct areas were automatically determined on the basis of total pixel intensity and area after segmentation of the red and green images, respectively. Automated measurements were compared with those made with a video camera-based image acquisition system that required manual tracing of lesion boundaries. RESULTS The spatial resolution of scanned brain images (approximately equal to 200 microns) was comparable to that of the camera-based system and provided sufficient detail to recognize infarct boundaries and neuroanatomical features. Scanner-based acquisition and analysis were faster than with the camera-based method. The green component image accurately distinguished infarcted from normal brain, and the red component image represented total brain dimensions. Infarct measurements obtained by the automated method correlated closely with those from conventional apparatus (R2 = .89, P < .001). Intraobserver reliability with the automated method (R2 = 1.00) was higher than with the conventional method (R2 = .77). CONCLUSIONS Infarct size after middle cerebral artery occlusion in the rat can be rapidly and reproducibly assessed with inexpensive scanning equipment and automated image analysis of TTC-stained brains.

Differences in vulnerability to permanent focal cerebral ischemia among 3 common mouse strains.

Background and Purpose Genetically engineered mice are used to study the role of single genes in cerebral ischemia, but inherent, strain-dependent differences in neuronal vulnerability may affect experimental end points. To examine this possibility, tissue injury resulting from focal ischemia and its relationship to cerebral hemodynamics were determined in 3 common mutant mouse strains. Methods Permanent middle cerebral artery ligation was performed in male C57BL/6J, Balb/C, and 129X1/SvJ mice. Mean arterial blood pressure, blood gases, basal and postischemic cortical blood flow ([14C]iodoantipyrine autoradiography and laser-Doppler flowmetry), posterior communicating artery patency, and infarct size were determined. Results Basal cortical blood flow did not differ among strains. Ten minutes after middle cerebral artery ligation, relative red cell flow in the ischemic cortex was 6% to 7% of preischemic flow in every strain. Despite similar hemodynamics, cortical infarcts in Balb/C mice were 3-fold larger than those in 129X1/SvJ and C57BL/6J mice; infarct size in the latter 2 strains was not significantly different. The posterior communicating artery was either poorly developed or absent in >90% of the Balb/C and C57BL/6J but in <50% of the 129X1/SvJ mice. Conclusions The extent of ischemic injury differed markedly between the 3 strains. The presence and patency of posterior communicating arteries, although variable among strains, did not affect preischemic or postischemic cortical blood flow or bear any relationship to ischemic injury. Therefore, intrinsic factors, other than hemodynamic variability, may contribute to the differences in ischemic vulnerability among strains. These findings underscore the importance of selecting genetically matched wild-type controls.

Differential Regulation of Thrombospondin-1 and Thrombospondin-2 After Focal Cerebral Ischemia/Reperfusion

Background and Purpose— Angiogenesis occurs after cerebral ischemia, and the extent of angiogenesis has been correlated with survival in stroke patients. However, postischemic angiogenesis is short-lived and may be completely terminated within a few weeks after ischemic insult. The molecular mechanism underlying the dissolution of postischemic angiogenic processes is poorly understood. Although the expression of angiogenic genes has been studied in ischemic stroke models, the activation of angiostatic genes after cerebral ischemia has not been investigated. Thrombospondin (TSP)-1 and TSP-2 are naturally occurring angiostatic factors, which inhibit angiogenesis in vivo. The aim of the present study was to explore the expression of TSP-1 and TSP-2 in relation to the evolution of angiogenic process in a focal ischemia model in rats. Methods— Rats underwent cortical ischemia in the middle cerebral artery territory for 60 minutes and reperfusion for up to 2 weeks. Northern and Western blot analysis were used to study the temporal profile of TSP-1 and TSP-2 expression at the mRNA and protein level, respectively. In situ hybridization and immunohistochemical studies were used to examine the spatial expression patterns. Double immunostaining was applied to define the cellular origins of TSP-1 and TSP-2. Results— A biphasic expression of TSP-1 was noted after ischemia, peaking at 1 and 72 hours. Endothelial cells in the leptomeninges were the only source of the first TSP-1 peak, whereas endothelial, glial, neuronal, and macrophage cells contributed to the second peak of TSP-1 expression. TSP-2 expression occurred much later and in a monophasic manner, peaking 2 weeks after ischemia. TSP-2 immunoreactivity was observed in endothelial, neuronal, and macrophage, but not glial, cells. TSP-1 was expressed before the peak of angiogenesis, whereas robust TSP-2 expression occurred at the peak of angiogenesis and continued into the period when angiogenesis had completely resolved. Conclusions— Robust expression of TSP-1 and TSP-2, 2 major angiostatic factors, was noted in the ischemic brain with different temporal expression profiles from different cellular origins. The expression of these angiostatic factors, especially TSP-2, likely contributes to the spontaneous resolution of postischemic angiogenesis. Further studies are needed to explore the molecular mechanisms that regulate the balance of angiogenic and angiostatic factors in the ischemic brain.

Effects of Lipopolysaccharide Priming on Acute Ischemic Brain Injury

BACKGROUND AND PURPOSE Infection has been implicated as a stroke risk factor. Activation and infiltration of polymorphonuclear neutrophils (PMNs) after cerebral ischemia may contribute to ischemic brain injury. This study was conducted to investigate how enhanced postischemic PMN infiltration by lipopolysaccharide (LPS) altered the acute ischemic outcomes. METHODS LPS (0.05 mg/kg SC) or vehicle was given to Long-Evans male rats 24 hours before ischemia. Focal cerebral ischemia was induced by temporary ligation of the right middle cerebral artery and both common carotid arteries for 45 minutes. Animals were killed 6 and 24 hours after reperfusion to determine the extent of PMN infiltration (myeloperoxidase assay), brain edema (wet-dry weight method), and vascular injury (fluorescein isothiocyanate-conjugated dextran extravasation). The infarct volumes were measured on the basis of TTC stain 24 hours after ischemia. RESULTS LPS had little effect on body temperature or peripheral white count but substantially enhanced PMN infiltration into the ischemic right middle cerebral artery cortex on the basis of myeloperoxidase activity (6 hours: control, 0 U/g; LPS, 0.186+/-0. 025 U/g; 24 hours: control, 0.185+/-0.025 U/g; LPS, 0.290+/-0.040 U/g; P<0.001) and morphological studies. The extent of vascular injury defined by the extravasation of fluorescein isothiocyanate-conjugated dextran into the ischemic tissue (6 hours: control, 3.11+/-0.41 microliter/mg protein; LPS, 0.48+/-0.16 microliter/mg protein; 24 hours: control, 1.77+/-0.23 microliter/mg protein; LPS, 0. 90+/-0.19 microliter/mg protein; P<0.001) and brain edema determined by the brain water content (6 hours: control, 84.77+/-1.63%; LPS, 82. 09+/-1.25%; 24 hours: control, 89.40+/-0.43%; LPS, 87.88+/-0.58%; P<0.01) were paradoxically reduced by LPS priming. LPS-primed rats also had smaller infarct volumes (control, 135+/-5 mm(3); LPS, 108+/-12 mm(3); P<0.05). CONCLUSIONS Enhanced postischemic PMN infiltration is anticipated to facilitate ischemic brain injury. Contrary to this expectation, results from the present study suggest that an increase in postischemic PMN infiltration after LPS priming was not detrimental. These findings challenge the notion that postischemic PMN infiltration is uniformly deleterious.

Multiple-Dose Mannitol Reduces Brain Water Content in a Rat Model of Cortical Infarction

BACKGROUND AND PURPOSE Repeated use of mannitol in the setting of ischemic infarction is a controversial and poorly defined therapeutic intervention. The purpose of this study was to examine the effects of repeated mannitol infusions on brain water content and tissue pressure in a well-defined rat model of focal ischemic stroke. METHODS Mannitol infusions (0.5, 1.5, or 2.5 g/kg) were given by intravenous bolus 4 or 24 hours after 90-minute transient cortical ischemia in the territory of the right middle cerebral artery in rats and every 4 hours thereafter for a total of 24 hours. Fluid replacement was limited to 0.5 mL i.v. isotonic saline administered immediately after each mannitol dose. Control rats received 0.5 mL i.v. saline at the same intervals and were otherwise under ad libitum conditions. Water contents (percent H2O) of whole hemispheres and of cortical biopsies were measured with the wet-dry method, and blood samples were analyzed for plasma osmolality and chemistries. In a subgroup of rats, tissue pressure was also measured within the hemisphere ipsilateral to the infarct. RESULTS Repeated mannitol infusions resulted in a dose-dependent increase in plasma osmolality and a dose-dependent decrease in the percent H2O of the ischemic middle cerebral artery cortex and ipsilateral hemisphere. In contrast, percent H2O of the contralateral cortex and hemisphere was significantly decreased only in the groups given the highest dose of mannitol (2.5 g/kg). Mannitol infusions at a dose of 1.5 g/kg begun 24 hours after reperfusion were also associated with a significant reduction of tissue pressure. CONCLUSIONS In a rat model of ischemic cortical infarction, repeated mannitol infusions resulted primarily in a decrease in the percent H2O of the infarct and ipsilateral hemisphere, as well as decreased tissue pressure.

Induction of secretory phospholipase A2 in reactive astrocytes in response to transient focal cerebral ischemia in the rat brain

Although mRNA expression of group IIA secretory phospholipase A2 (sPLA2‐IIA) has been implicated in responses to injury in the CNS, information on protein expression remains unclear. In this study, we investigated temporal and spatial expression of sPLA2‐IIA mRNA and immunoreactivity in transient focal cerebral ischemia induced in rats by occlusion of the middle cerebral artery. Northern blot analysis showed a biphasic increase in sPLA2‐IIA mRNA expression following 60‐min of ischemia–reperfusion: an early phase at 30 min and a second increase at a late phase ranging from 12 h to 14 days. In situ hybridization localized the early‐phase increase in sPLA2‐IIA mRNA to the affected ischemic cortex and the late‐phase increase to the penumbral area. Besides sPLA2‐IIA mRNA, glial fibrillary acidic protein (GFAP) and cyclo‐oxygenase‐2 mRNAs, but not cytosolic PLA2, also showed an increase in the penumbral area at 3 days after ischemia–reperfusion. Immunohistochemistry of sPLA2‐IIA indicated positive cells in the penumbral area similar to the GFAP‐positive astrocytes but different from the isolectin B4‐positive microglial cells. Confocal microscopy further confirmed immunoreactivity of sPLA2‐IIA in reactive astrocytes but not in microglial cells. Taken together, these results demonstrate for the first time an up‐regulation of the inflammatory sPLA2‐IIA in reactive astrocytes in response to cerebral ischemia–reperfusion.

Absolute measurements of water content using magnetic resonance imaging: Preliminary findings in an in vivo focal ischemic rat model

Using a magnetic resonance (MR) imaging method, absolute measurements of in vivo brain water content were obtained in 15 male Long Evans rats that underwent a 90‐min focal cerebral ischemia. A strong linear relationship (r = 0.80) with a slope of 1 was observed when correlating MR‐measured water content to that obtained with the ex vivo wet/dry measurements. This effective spin‐density‐based method is the first‐reported in vivo absolute quantification of brain tissue water content associated with a pathophysiological state and preliminary findings suggest that a noninvasive measurement of brain water content can be obtained with MRI. Magn Reson Med 43:146–150, 2000.

Effects of Fluid Management on Edema Volume and Midline Shift in a Rat Model of Ischemic Stroke

BACKGROUND AND PURPOSE The purpose of this study was to investigate the effects of fluid management on brain water content (BW) and midline shift (MLS) after a focal cerebral ischemic insult. METHODS A suture model was used to induce focal cerebral ischemia for 90 minutes (n=44). The rats were randomly assigned to 3 groups 2. 5 hours after reperfusion: dehydration (n=24), control (n=8), or hydration (n=12). BW was obtained with the wet-dry weight method 24 hours after middle cerebral artery (MCA) occlusion. In addition, MRI were obtained (n=31) 24 hours after the onset of ischemia so that the ratio of hemispheric volumes ipsilateral (IH) and contralateral (CH) to the infarct and the extent of MLS could be obtained. RESULTS Across the range from moderate dehydration to intravascular volume expansion with isotonic saline, BW of the IH increased linearly as a function of change in body weight (r(2)=0.89), whereas few changes in relation to body weight were observed in CH, indicating a preferential effect of fluid management on the infarcted hemisphere. Furthermore, the hemispheric volume ratio (IH/CH) and MLS also increased in relation to changes in body weight. However, paradoxical increases in BW, IH/CH, and extent of MLS were observed in comparison with controls when severe dehydration was produced with high-dose mannitol. CONCLUSIONS Changes in ischemic BW by fluid management correlated closely with changes in body weight except when high-dose mannitol was used. Mannitol, as a dehydrating agent, may be associated with bimodal effects, with a high dose aggravating ischemic BW.

An Absolute Measurement of Brain Water Content Using Magnetic Resonance Imaging in Two Focal Cerebral Ischemic Rat Models

Magnetic resonance imaging (MRI) was utilized to obtain absolute estimates of regional brain water content (W), and results were compared with those obtained with conventional wet/dry measurements. In total, 31 male Long-Evans rats were studied and divided into two groups based on the surgical procedures used to induce cerebral focal ischemia: suture (n = 18) and three-vessel ligation (TVL; n = 13) groups. Both relative spin density and T1 were extracted from the acquired MR images. After correcting for radiofrequency field inhomogeneities, T2* signal decay, and temperature effects, in vivo regional brain water content, in absolute terms, was obtained by normalizing the measured relative brain spin density of animals to that of a water phantom. A highly linear relationship between MR-estimated brain water content based on the normalized spin density and wet/dry measurements was obtained with slopes of 0.989 and 0.986 for the suture (r = 0.79) and TVL (r = 0.83) groups, respectively. Except for the normal subcortex of the TVL group (P < 0.02) and the normal hemisphere of the suture group (P < 0.003), no significant differences were observed between MR-estimated and wet/dry measurements of brain water content. In addition, a highly linear relationship between MR-measured R1 (= 1/T1) and 1/W of wet/dry measurements was obtained. However, slopes of the linear regression lines in the two groups were significantly different (P < 0.02), indicating that different R1 values were associated with the same water content depending on the model. These results show that an absolute measurement of in vivo regional brain water content can be obtained with MRI and potentially serves as a noninvasive means to monitor different therapeutic interventions for the management of brain edema subsequent to stroke and head trauma.