Ischemic Core Area Research Articles

Role of sodium/hydrogen exchanger isoform 1 in microglial activation and proinflammatory responses in ischemic brains

Our recent study reveals that Na⁺/H⁺ exchanger isoform 1 (NHE-1) mediates H⁺ extrusion during "respiratory bursting", which is important for microglial activation. In the present study, we further investigated whether NHE-1 plays a role in proinflammatory activation of microglia in vivo using a mouse model of transient focal cerebral ischemia and reperfusion (I/R). Activated microglial cells were identified by their expression of two microglial marker proteins (CD11b and Iba1) as well as by their transformation from a "ramified" to an "amoeboid" morphology. An immediate increase in activated microglial numbers was detected in the ipsilateral ischemic core area of NHE-1⁺/⁺ brains at 1 hour (h) I/1 h R, which gradually decreased during 6-24 h I/R. This was followed by a sharp rise in microglial activation in the peri-infarct area and an increase in proinflammatory cytokine formation at 3 day after I/R. Interestingly, HOE 642 (a potent NHE-1 inhibitor) -treated or NHE-1 heterozygous (NHE-1⁺/⁻) mice exhibited less microglia activation, less NADPH oxidase activation, or a reduced proinflammatory response at 3-7 day after I/R. Blocking NHE-1 activity also significantly decreased microglial phagocytosis in vitro. In contrast, astrogliosis formation in the peri-infarct area was not affected by NHE-1 inhibition. Taken together, our results demonstrate that NHE-1 protein was abundantly expressed in activated microglia and astrocytes. NHE-1 inhibition reduced microglial proinflammatory activation following ischemia.

MRI of stroke using hyperpolarized 129Xe

Because there is no background signal from xenon in biological tissue, and because inhaled xenon is delivered to the brain by blood flow, we would expect a perfusion deficit, such as is seen in stroke, to reduce the xenon concentration in the region of the deficit. Thermal polarization yields negligible xenon signal relative to hyperpolarized xenon; therefore, hyperpolarized xenon can be used as a tracer of cerebral blood flow. Using a rat permanent right middle cerebral artery occlusion model, we demonstrated that hyperpolarized (129)Xe MRI is able to detect, in vivo, the hypoperfused area of focal cerebral ischemia, that is the ischemic core area of stroke. To the best of our knowledge, this is the first time that hyperpolarized (129)Xe MRI has been used to explore normal and abnormal cerebral perfusion. Our study shows a novel application of hyperpolarized (129)Xe MRI for imaging stroke, and further demonstrates its capacity to serve as a complementary tool to proton MRI for the study of the pathophysiology during brain hypoperfusion.

Protective role of hematopoietic prostaglandin D synthase in transient focal cerebral ischemia in mice

Cerebral ischemia/reperfusion injury is characterized by the development of inflammatory response, in which vascular macrophages and endogenous microglia are involved. Recent studies showed marked induction of hematopoietic prostaglandin D synthase (HPGDS) after ischemic/reperfusion injury and its localization in microglia, but the molecular mechanism(s) of HPGDS actions in cerebral ischemia is not clear. To clarify the role of HPGDS in cerebral ischemia, C57BL/6 mice and bone marrow chimera mice with cerebral ischemia/reperfusion injury were treated with (4-benzhydryloxy-(1) {3-(1H-tetrazol-5-yl)-propyl}piperidine (HQL-79), a specific inhibitor of HPGDS. The bone marrow chimera mice exhibit expression of enhanced green fluorescent protein (EGFP) in bone marrow/blood-derived monocytes/macrophages. Mice were subjected to ischemia/reperfusion and either treated with HQL-79 (n=44) or vehicle (n=44). Brain sections prepared at 72 h and 7 days after reperfusion were analyzed for neuronal nuclei (NeuN), HPGDS, ionized calcium-binding adapter molecule 1 (Iba1), inducible NO synthase (iNOS), nitrotyrosine, nuclear factor kappa B (NF-kB) and cyclooxygenase-2 (COX-2). The mortality rate (80%) and infarct size were larger in HQL-79- than vehicle-treated mice (58.7±8.5 versus 45.2±4.9 mm3; mean±SEM, P<0.0001) at 7 days after reperfusion. HQL-79 reduced NeuN expression in the transition area and Iba1 expression (P<0.0001) in the ischemic peri- and penumbra area, but increased COX-2 (P<0.05) and NF-kB expression (P<0.05) in ischemic penumbra and increased formation of nitrotyrosine (P<0.0001) and iNOS (P<0.0001) in the ischemic core area at 72 h and 7 days after reperfusion. In EGFP chimera mice, HQL-79 increased the migration of Iba1/EGFP-positive bone marrow-derived monocytes/macrophages, and simultaneously upregulated iNOS expression in the ischemic core area (P<0.0001), but increased intrinsic microglia/macrophages in ischemic peri-area and penumbra (P<0.0001) at 72 h and 7 days after reperfusion, suggesting involvement of monocytes/macrophages in HQL-79-induced expansion of ischemic injury. Our results demonstrated that the neuroprotective effects of HPGDS in our model are mediated by suppression of activation and infiltration of inflammatory cells.

Hyperglycemia-enhanced ischemic brain damage in mutant manganese SOD mice is associated with suppression of HIF-1α

Both preischemic hyperglycemia and reduction of manganese superoxide dismutase activity are known to enhance neuronal death induced by transient cerebral ischemia. Transcriptional factor hypoxia-inducible factor 1 (HIF-1) regulates multiple downstream genes that modulate cell metabolism, survival, death, angiogenesis, hematopoiesis, and other functions. The objectives of this study were to determine (i) whether hyperglycemia is able to increase ischemic brain damage in mutant manganese superoxide dismutase (SOD2) mice and (ii) whether the reduction of SOD2 activity has a profound effect on HIF-1 protein expression under hyperglycemic ischemic condition. Both wild type and mutant SOD deficient (SOD2 −/+) mice were induced to hyperglycemia 30 min before induction of a 30-min transient middle cerebral artery occlusion (tMCAO). Brains were extracted after 5 and 24 h of reperfusion for immunohistochemistry and Western blot analyses. The results showed that preischemic hyperglycemia significantly increased infarct volume in SOD2 −/+mice and that HIF-1α protein levels were significantly reduced in ischemic core area at 5- and 24-h of reperfusion in hyperglycemic SOD2 −/+ mice. However, the HIF-1α protein levels were not significantly decreased in hyperglycemic wild type animals subjected to stroke. The results suggest that the increased brain damage observed in hyperglycemic SOD2 −/+ mice is associated with HIF-1α suppression, while hyperglycemia per se does not seem to exert its detrimental effects on ischemic brain via modulating HIF-1 pathway.

Can exogenous stem cells improve outcome after experimental stroke? The challenge of combined MRI imaging of stem cell dynamics, cell differentiation and functional outcome

Conventional therapies of stroke have so far demonstrated limited success. Cerebroprotective drugs have been extensi-vely and rather successfully investigated in animal experiments. In the clinical environment, however, all drugs have failed so far. Throm-bo-lysis is the only strategy with successful translation from experimental into clinical neurology. Experimental and clinical research on thrombolysis of stroke has extensively documented that it will prevent further expansion of the ischemic lesion by stabilizing the penumbral tissue but it will not reverse the damage of the early irreversibly damaged ischemic core area. The recent breath-taking developments in stem cell biology have motivated the evaluation of the therapeutic potential of endogenous or implan-ted stem cells as a cell replacement therapy for stroke. Arvidsson et al. 1 demonstrated a significant activation of neurogenesis in the subventricular zone of ischemic rats while the lab of Helen Hodges 2 reported on migration of implanted ES cells towards the lesion periphery in ischemic rats, followed by outcome improve-ments relative to animals without cell implantations. Using fMRI two groups investigated the sponta-neous functional improvement and activation potential of the somatosensory cortex of rats following induction of stroke reporting a transient re-organization process, during which the representation area was shifted to the contralateral, undamaged hemisphere during a transition period 3, 4. While some investigators looked at histological evolution of the lesion after treatment with stem cell implantation, no study focussed on the mechanisms of behavior improvements in these treated situations: restitution of function of the original repre-sen-tation area or plasticity dependent re-organization, enhanced by the stem cell inter-action with the host organ. Such investigations present an enormous challenge on the design and execution of a complex experimental protocol. On the one hand, the evolution of the lesion must be characterized in parallel with the fate of the implanted stem cells (migrational dynamics to the target zone; cell specific differentiation). On the other hand, a potential therapeutical success must be evaluated in its temporal development. Finally, mechanisms explaining a potential outcome improvement are of great interest. Most recent investigations are exploring the potential contribution of molecular imaging techniques and have reported the in vivo monitoring of stem cell migration in rat 5 and mouse brain after stroke 6. The present contribution will demonstrate the possibilities (and limits) of non-invasive imaging modalities for the characterization of the lesion and the (implanted) cell mobility, while the functional fate (e.g. differentiation status) is described by way of immuno-histochemistry. Parallel investigations on the functional deficit and outcome improvement, respectively, are presented. Mechanisms responsible for a functional improvement (statial reorganization vs recovery of primary representation field) can be studied by longitudinal fMRI studies of anesthetized animals, allowing the localization of activated brain areas.

Cerebral tissue oxygenation in reversible focal ischemia in rats: multi-site EPR oximetry measurements

Multi-site electron paramagnetic resonance (EPR) oximetry was used in vivo to measure the partial pressure of oxygen (pO2) in reversible focal ischemia in rats. The cerebral tissue pO2 was measured simultaneously and continuously at two sites on the ischemic side and one on the normal side of the brain in the same animal prior to and at several time points after ischemia and reperfusion. The O2 at the three different sites in brain was stable over 30 min of baseline measurements. During the first 120 min of ischemia, statistically significant decreases in brain pO2 from baseline were consistently observed in the ischemic core and perifocal area. The mean values varied during the 120 min of ischemia. Reperfusion resulted in an immediate increase in pO2, but there were no significant differences between the sites over time. The result of this study seems promising for the study of ischemia and reperfusion. It appears that the technique can provide information on the pO2 under the experimental conditions needed for such a study. The levels of pO2 that occurred in these experiments are readily resolvable by multi-site EPR oximetry. In addition, the ability simultaneously to measure the pO2 in several sites provides important additional information that should help to differentiate between changes in the pO2 due toglobal or local mechanisms.

Glucosylceramide synthase activity and ceramide levels are modulated during cerebral ischemia after ischemic preconditioning.

After 24-hour middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats, brain ceramide level increased from baseline reached 595% (ischemic core) and 460% (perifocal/penumbral areas); brain glucosylceramide synthase (GCS) activities in these areas simultaneously decreased by 70% and 50%, respectively. Ten-minute MCAO preconditioning significantly attenuated 24-hour MCAO-induced ceramide accumulation by 40% to 60% in ischemic core and perifocal areas, and GCS activities improved by 60% to 70% in both areas. Thus, potentially toxic levels of brain ceramide induced by MCAO were attenuated to intermediate levels in preconditioned animals; brain GCS activity was relatively preserved. In ischemic tolerance, GCS appears to modulate otherwise high levels of brain ceramide.

Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia

Brain ischemia induces a marked response of resident microglia and hematopoietic cells including monocytes/macrophages. The present study was designed to assess the distribution of microglia/macrophages in cerebral ischemia using bone marrow chimera mice known to express enhanced green fluorescent protein (EGFP). At 24 h after middle cerebral artery occlusion (MCAO), many round-shaped EGFP-positive cells migrated to the ischemic core and peri-infarct area. At 48–72 h after MCAO, irregular round- or oval-shaped EGFP/ionized calcium-binding adapter molecule 1 (Iba 1)-positive cells increased in the transition zone, while many amoeboid-shaped or large-cell-body EGFP/Iba 1-positive cells were increased in number in the innermost area of ischemia. At 7 days after MCAO, many process-bearing ramified shaped EGFP/Iba 1-positive cells were detected in the transition to the peri-infarct area, while phagocytic cells were distributed in the transition to the core area of the infarction. The distribution of these morphologically variable EGFP/Iba 1-positive cells was similar up to 14 days from MCAO. The present study directly showed the migration and distribution of bone marrow-derived monocytes/macrophages and the relationship between resident microglia and infiltrated hematogenous element in ischemic mouse brain. It is important to study the distribution of intrinsic and extrinsic microglia/macrophage in ischemic brain, since such findings may allow the design of appropriate gene-delivery system using exogenous microglia/macrophages to the ischemic brain area.

Induction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in rat brain after focal ischemia/reperfusion.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, has been recently identified to be involved in the initiation of neuronal apoptosis. To investigate the serial changes and cellular localization of GAPDH expression, and its role in ischemia/reperfusion-induced neuronal apoptosis, the authors analyzed immunohistochemically brain areas of rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Nuclear overexpression of GAPDH was noted in the ischemic core area after 2 hours of MCAO without reperfusion. During the subsequent reperfusion, nuclear accumulation of GAPDH in this area decreased in a time-dependent manner. However, cytoplasmic and nuclear GAPDH immunoreactivity was detected in neurons of the penumbra area of the parietal cortex, in rats subjected to 2-hour MCAO followed by 3-hour reperfusion. The increase of nuclear GAPDH immunoreactivity was persistently noted up to 48 hours of reperfusion, whereas cytoplasmic immunoreactivity correlated inversely with the duration of reperfusion. Moreover, double staining revealed colocalization of nuclear GAPDH and TUNEL in the penumbra area. The authors' study demonstrated that overexpression of GAPDH and nuclear translocation occurred in both the ischemic core and penumbra area soon after focal ischemia. These processes could be viewed as an early marker of ischemia/reperfusion-induced apoptotic neuronal death. The results suggest that GAPDH may play a critical role in the progression and spread of ischemic neuronal damage.

DNA fragmentation in ischemic core and penumbra in focal cerebral ischemia in rats

Although apoptotic cell death has been suggested to be involved in ischemic injury of the brain, the precise mechanisms of ischemic neuronal cell death are unknown. Here, we examined the biochemical feature of apoptosis (i.e. DNA fragmentation) in male spontaneously hypertensive rats (5–7 months old) subjected to photothrombotic distal middle cerebral artery (MCA) occlusion. After MCA occlusion, the brain was cut in a cryostat to produce a standard coronal block and samples were dissected from the regions corresponding to the ischemic core, penumbra and contralateral control areas. Changes in cerebral blood flow (CBF) were monitored at 1 mm posterior and 2–4 mm lateral to the bregma by means of a laser-Doppler flowmetry. After MCA occlusion, CBF was decreased to 72±18 (±S.D.), 50±14, and 35±11% of the control values at 2, 3, and 4 mm from the midline, respectively. DNA fragmentation characteristics of apoptosis were examined in these samples by conventional and pulse-field gel electrophoresis. On the conventional gel electrophoresis, nucleosomal DNA fragmentation was detected in the penumbral zone at 6 h after MCA occlusion. Large DNA fragments of 50 and 20 kbp were detected in the penumbral zone and also in the ischemic core region at 3 h after distal MCA occlusion. The large DNA fragments seen on the pulse-field gel elecrophoresis were further degraded to small DNA fragments at 6 h after MCA occlusion in the penumbral zone but not in the core regions. The evolving DNA fragmentation was observed between 3 and 6 h after the onset of brain ischemia in the penumbra, suggesting that apoptosis may contribute to the development of ischemic infarction.

Cerebral Blood Flow, Glucose Metabolism and TUNEL-Positive Cells in the Development of Ischemia

Few in vivo studies were available about the relation between cerebral blood flow, glucose metabolism and the appearance of apoptotic cells in the development of cerebral infarct. To investigate this, we measured local cerebral blood flow (lCBF), local cerebral metabolic rate in glucose (lCMRglc), and histopathology in transient focal cerebral ischemia in the rat. A unilateral middle cerebral artery occlusion (MCAO) was induced for 2 h in Wistar-ST rats (n = 42). A histopathological study with hematoxylin-eosin staining and the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method was performed. lCBF was measured by means of the ¹⁴C-iodoantipyrine autoradiography technique during MCAO (n = 6), and 1, 22 and 70 h after reperfusion. lCMRglc was also measured by autoradiography with ¹⁴C-2-deoxyglucose in the animals 22 h after reperfusion. These parameters were assessed in each region of interest: the ischemic core, boundary zones (BZ-I and BZ-II) and remote area. The boundary zones were defined as the area based on TUNEL positivity (more than 5/field) at 22 h after reperfusion (BZ-I) and at 70 h after reperfusion (BZ-II). In the BZ-I, lCBF was decreased to 18% of the control during MCAO, and lCBF and lCMRglc showed 44 and 62% of the control, respectively, 22 h after reperfusion. In this area, TUNEL-positive cells increased at 22 h, then markedly decreased 70 h after reperfusion. In the BZ-II, lCBF decreased to 39% of the control during MCAO, then returned to about 90% of the control 22 h after reperfusion. lCMRglc was maintained near its normal range (82% of the control) 22 h after reperfusion. Histopathology of BZ-II was normal 22 h after reperfusion. The TUNEL positivity of neurons in our study was assumed to be a marker of apoptotic cells. Our data suggested that the apoptotic process plays an important role in the maturation of a cerebral infarct. Both lCBF and lCMRglc were maintained with only a mild reduction in the predisposing phase of apoptosis, suggesting that sufficient blood supply and glucose metabolism are required to promote the process of apoptosis.

CORE AND PENUMBRAL NITRIC OXIDE SYNTHASE ACTIVITY DURING FOCAL CEREBRAL ISCHEMIA AND REPERFUSION IN RATS

S340 Introduction: Although the neurotoxic effects of nitric oxide during cerebral ischemia are recognized, [1] a differential effect of the severity of ischemia on cerebral nitric oxide synthase (NOS) activity has not been described. We measured NOS activity in ischemic core and penumbral brain areas after middle cerebral artery occlusion (MCAo) and reperfusion in rats. Methods: The protocol was approved by the animal care committee. Physiologic parameters were maintained within normal limits (cranial temperature-37[degree sign]C). During isoflurane anesthesia, MCAo was achieved by advancing a suture into the middle cerebral artery for 90-min. NOS activity (14) C-arginine[right arrow]14 C-citrulline) was measured in core and penumbral brain regions [2] in the ischemic hemisphere, and corresponding areas in the nonischemic hemisphere for three groups, before and after addition of NOS cofactors (tetrahydrobiopterin, flavin adenine dinucleotide, flavin mononucleotide): Controls (n=10)-after immediate decapitation (no MCAo); MCAo (n=11)-90-min of MCAo and no reperfusion; MCAo/R (n=9)-90-min of MCAo and 30-min of reperfusion. The data were evaluated by ANOVA. Results: Addition of cofactors increased NOS activity in all areas for all groups versus the corresponding area without cofactors. In all groups, core NOS activity was greater than penumbral NOS activity (p<0.05). In the ischemic core, NOS activity was less in the MCAo/R group than the MCAo group. Moreover, NOS activity in the core was significantly less than corresponding tissue from the right hemisphere (see Table 1).Table 1: NOS activity, without and with cofactors for the ischemic core (I-Core) and penumbral (I-Pen) areas, and for corresponding areas in the non-ischemic hemisphere (N-Core, N-Pen). The units are fmol[center dot]mg of prot-1[center dot]min-1 (mean +/- SEM). *p<0.05 versus the corresponding penumbral area. sup [dagger] p<0.05 versus the non-ischemic core. sup [double dagger] p<0.05 versus the MCAo group. sup & p<0.05 versus the corresponding area without cofactors.Discussion: The data demonstrate higher core than penumbral NOS activity before MCAo, during occlusion, and during early reperfusion. These differences suggest that NOS activity may act to define vulnerable areas of the brain to ischemia.

Regional cerebral blood flow during and after 2 hours of middle cerebral artery occlusion in the rat.

In this study we explored if the secondary bioenergetic failure, which occurs a few hours after recirculation, following transient middle cerebral artery occlusion (MCAO) in rats, is caused by a compromised reflow. We induced 2 hours of MCAO and measured CBF at the end of the ischemia, as well as 15 minutes, 1, 2, and 4 hours after the start of recirculation, using autoradiographic or tissue sampling 14C-iodoantipyrine techniques. After 2 hours of MCAO, the autoradiographically measured CBF in the ischemic core areas was reduced to 3 to 5% of contralateral values. The reduction in CBF was less in neighboring, penumbral areas. After recirculation, flow already normalized in core tissues after 15 minutes, and remained close to normal for the 4 hours recirculation period studied. However, in penumbral tissues, recovery CBF values were usually below normal. The results show that tissues that are heavily compromised by the 2-hour period of ischemia and are destined to incur infarction, show a "relative hyperemia" during recirculation. In fact, some areas of the previously densely ischemic tissue showed overt hyperperfusion. This finding raises the question whether the relative or absolute hyperemia reflects events that are pathogenetically important. Because drugs that clearly ameliorate the final damage incurred fail to alter the relative hyperperfusion of previously ischemic tissues, it is concluded that vascular events in the reperfusion period do not play a major role in causing the final damage.

Extracellular potassium in a neocortical core area after transient focal ischemia.

Occlusion of the middle cerebral artery (MCAO) results in bioenergetic failure in the densely ischemic core areas. During reperfusion, transient recovery of the bioenergetic state is followed by secondary deterioration. In this study, we recorded the extracellular potassium concentrations in the cortical core during 2 hours of MCAO, as well as during recovery. One group of animals with recirculation periods of 6 to 8 hours was given the free radical spin trap alpha-phenyl-N-tert-butyl nitrone (PBN). The experiments were performed on adult male Wistar rats (305 to 335 g). The right MCA was occluded by an intraluminal filament technique. For [K+]e measurements a craniotomy was made over the right cortex, and an ion-sensitive microelectrode was lowered into the ischemic focus. Recording of [K+]e was continued for 2 hours. After 48 hours of reperfusion, infarction size was estimated with 2,3,5-triphenyltetrazolium chloride. During MCA occlusion, [K+]e rose to approximately 60 mmol/L. However, several animals showed transient (and partial) periods of repolarization accompanied by a decrease in [K+]e. Immediately on reperfusion, the [K+]e started to recover and reached baseline levels (2.5 mmol/L) within 3 to 5 minutes. During the first 6 hours of recovery, [K+]e was stable at about 2.5 mmol/L, but after this period a moderate increase in the [K+]e was observed. This was not observed in animals injected with PBN 1 hour after reperfusion. The data suggest that delayed cell membrane dysfunction, as reflected in a rise in [K+]e, occurs after about 6 hours of reperfusion and that treatment with PBN in a single dose ameliorates or delays such deterioration of plasma membrane function.

Appearance of immunoglobulin G and complement factor C3 in the striatum after transient focal ischemia in the rat

The pathophysiological feature of brain ischemia-infarct was investigated using immunohistochemistry and Gallyas' silver staining after transient ischemia of the middle cerebral artery (MCA) in the rat. A very strong IgG infiltration with clear-cut borders was detected at one to 3 days in ischemic core areas (lateral striatum and adjacent cortex) that fell into porencephaly later. Complement factor C3 (C3) immunoreactivity (IR) appeared similarly while albumin IR more diffusely. Microglial activation could be observed at 1 day while rounded leukocyte-like elements at 3 days after reperfusion. Data suggest that the early appearance of the IgG/C3 IR bears particular importance after transient MCA ischemia as it could predict the ensuing porencephaly in the chronic stage.

Reductions in focal ischemic infarctions elicited from cerebellar fastigial nucleus do not result from elevations in cerebral blood flow.

To determine whether the neuroprotection elicited from electrical stimulation of the cerebellar fastigial nucleus (FN) is attributable to the elevation in regional cerebral blood flow (rCBF), we compared the effects in spontaneously hypertensive rats of stimulation of the rostral ventrolateral medulla (RVL) or FN on (a) a focal ischemic lesion produced by middle cerebral artery (MCA) occlusion, and (b) the changes in rCBF, measured by laser-Doppler flowmetry for 1.5 h, over regions corresponding to the ischemic core (parietal cortex), penumbra (occipital cortex), and nonischemic area (contralateral parietal cortex). Stimulation of FN for 1 h following MCA occlusion reduced infarction 24 h later by 52%. Stimulation of RVL was ineffective. Changes in the lesion were confined to the penumbra. FN and RVL stimulation comparably and significantly increased rCBF up to 185% in unlesioned animals. Following MCA occlusion, stimulation of FN or RVL and hypercarbia failed to elevate rCBF in the ischemic area but did so in the nonischemic area, even though in the same animals only FN stimulation reduced infarction 24 h later. We conclude that (a) the neuroprotection elicited from FN is not the result of an increase in rCBF but results from another mechanism, possibly reduction of metabolism in penumbra, and (b) the pathways mediating central neurogenic vasodilation and neuroprotection are, in part, distinct.

CO2 reactivity and heterogeneity of cerebral blood flow in ischemic, border zone, and normal cortex

Regional arterial CO2 tension (PaCO2) reactivity of cerebral blood flow (CBF) and the effect of PaCO2 on the spatial and temporal heterogeneity of CBF were investigated by using autoradiographically determined CBF in the rat middle cerebral artery occlusion model after a 2-h period under pentobarbital anesthesia to clarify the relation between PaCO2 reactivity, CBF heterogeneity, and the temporal cycling of CBF. PaCO2 was adjusted to one of four levels. CBF was determined in four cortical areas and white matter using the tissue fractionation of [14C]iodoantipyrine [( 14C]IAP) in combination with vessel mapping using in vivo 4% thioflavine S. Specific PaCO2 reactivity and CBF were normal in the nonischemic cortex, normal, although slightly depressed, in the border zone far from the ischemic core area, and depressed in the border zone adjacent to the ischemic core area (P less than 0.001) and the ischemic core (P less than 0.001). In normocapnic and hypocapnic animals, CBF heterogeneity in the form of regularly spaced CBF columns perpendicular to the cortical surface was observed in the nonischemic hemisphere but was absent in the ischemic core area. In hypercapnic rats, flow columns were present in the ischemic core areas and border zones but were absent on the nonischemic side. There was a highly significant interaction (P less than 0.0001) in observer-determined heterogeneity grades between PaCO2 level and each of three areas, normal, border zone, and ischemic core. In normal cortex, comparison of the thioflavine S-stained vessels with the flow columns provided evidence supporting the concept of capillary recruitment and cycling as a mode of normal cerebral blood flow control. The presence of flow columns in severely ischemic areas in hypercapnic animals indicates that a short period of high PaCO2 transiently augments microregional flow and could enhance the delivery of a therapeutic agent to these microregions of the ischemic core. The regional analysis of PaCO2 reactivity suggests an index of future tissue viability.