Nikita Derugin

Rat forebrain neurogenesis and striatal neuron replacement after focal stroke

The persistence of neurogenesis in the forebrain subventricular zone (SVZ) of adult mammals suggests that the mature brain maintains the potential for neuronal replacement after injury. We examined whether focal ischemic injury in adult rat would increase SVZ neurogenesis and direct migration and neuronal differentiation of endogenous precursors in damaged regions. Focal stroke was induced in adult rats by 90‐minute right middle cerebral artery occlusion (tMCAO). Cell proliferation and neurogenesis were assessed with bromodeoxyuridine (BrdU) labeling and immunostaining for cell type‐specific markers. Brains examined 10–21 days after stroke showed markedly increased SVZ neurogenesis and chains of neuroblasts extending from the SVZ to the peri‐infarct striatum. Many BrdU‐labeled cells persisted in the striatum and cortex adjacent to infarcts, but at 35 days after tMCAO only BrdU‐labeled cells in the neostriatum expressed neuronal markers. Newly generated cells in the injured neostriatum expressed markers of medium spiny neurons, which characterize most neostriatal neurons lost after tMCAO. These findings indicate that focal ischemic injury increases SVZ neurogenesis and directs neuroblast migration to sites of damage. Moreover, neuroblasts in the injured neostriatum appear to differentiate into a region‐appropriate phenotype, which suggests that the mature brain is capable of replacing some neurons lost after ischemic injury.

Microglial Cells Contribute to Endogenous Brain Defenses after Acute Neonatal Focal Stroke

Macrophages are viewed as amplifiers of ischemic brain injury, but the origin of injury-producing macrophages is poorly defined. The role of resident brain macrophages—microglial cells—in stroke remains controversial. To determine whether microglial cells exert injurious effects after neonatal focal stroke, we selectively depleted these cells with intracerebral injection of liposome-encapsulated clodronate before transient middle cerebral artery occlusion in postnatal day 7 rats. Phagocytosis of apoptotic neurons by activated microglia was poor in animals with unmanipulated microglia, and depletion of these cells did not increase the number of apoptotic neurons. Lack of microglia increased the brain levels of several cytokines and chemokines already elevated by ischemia–reperfusion, and also increased the severity and volume of injury, suggesting that microglial cells contribute to endogenous protection during the subacute injury phase. Then, to determine whether accumulation of reactive oxygen species in microglia adversely affects phagocytosis of dying neurons and contributes to injury, we delivered reduced glutathione (GSH) into microglia, again using liposomes. Remarkably, pharmacologically increased intracellular GSH concentrations in microglia induced superoxide accumulation in lipid rafts in these cells, further increased the brain levels of macrophage chemoattractants, and exacerbated injury. Together, these data show that microglia are part of the endogenous defense mechanisms and that, while antioxidants can protect the injured neonatal brain, high levels of reducing equivalents in activated microglia, GSH, trigger superoxide production, favor the reorganization of lipids, amplify local inflammation and exacerbate injury.

Minocycline confers early but transient protection in the immature brain following focal cerebral ischemia–reperfusion

The incidence of neonatal stroke is high and currently there are no strategies to protect the neonatal brain from stroke or reduce the sequelae. Agents capable of modifying inflammatory processes hold promise. We set out to determine whether delayed administration of one such agent, minocycline, protects the immature brain in a model of transient middle cerebral artery (MCA) occlusion in 7-day-old rat pups. Injury volume in minocycline (45 mg/kg/dose, beginning at 2 h after MCA occlusion) and vehicle-treated pups was determined 24 h and 7 days after onset of reperfusion. Accumulation of activated microglia/macrophages, phosphorylation of mitogen-activated protein kinase (MAPK) p38 in the brain, and concentrations of inflammatory mediators in plasma and brain were determined at 24 h. Minocycline significantly reduced the volume of injury at 24 h but not 7 days after transient MCA occlusion. The beneficial effect of minocycline acutely after reperfusion was not associated with changed ED1 phenotype, nor was the pattern of MAPK p38 phosphorylation altered. Minocycline reduced accumulation of IL-1β and CINC-1 in the systemic circulation but failed to affect the increased levels of IL-1β, IL-18, MCP-1 or CINC-1 in the injured brain tissue. Therefore, minocycline provides early but transient protection, which is largely independent of microglial activation or activation of the MAPK p38 pathway.

Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat

The immaturity of the CNS at birth greatly affects injury after stroke but the contribution of the blood–brain barrier (BBB) to the differential response to stroke in adults and neonates is poorly understood. We asked whether the structure and function of the BBB is disrupted differently in neonatal and adult rats by transient middle cerebral artery occlusion. In adult rats, albumin leakage into injured regions was markedly increased during 2–24 h reperfusion but leakage remained low in the neonates. Functional assays employing intravascular tracers in the neonates showed that BBB permeability to both large (70 kDa dextran) and small (3 kDa dextran), gadolinium (III)-diethyltriaminepentaacetic acid tracers remained largely undisturbed 24 h after reperfusion. The profoundly different functional integrity of the BBB was associated with the largely nonoverlapping patterns of regulated genes in endothelial cells purified from injured and uninjured adult and neonatal brain at 24 h (endothelial transcriptome, 31,042 total probe sets). Within significantly regulated 1266 probe sets in injured adults and 361 probe sets in neonates, changes in the gene expression of the basal lamina components, adhesion molecules, the tight junction protein occludin, and matrix metalloproteinase-9 were among the key differences. The protein expression of collagen-IV, laminin, claudin-5, occludin, and zonula occludens protein 1 was also better preserved in neonatal rats. Neutrophil infiltration remained low in acutely injured neonates but neutralization of cytokine-induced neutrophil chemoattractant-1 in the systemic circulation enhanced neutrophil infiltration, BBB permeability, and injury. The markedly more integrant BBB in neonatal brain than in adult brain after acute stroke may have major implications for the treatment of neonatal stroke.

Macrophages are comprised of resident brain microglia not infiltrating peripheral monocytes acutely after neonatal stroke.

Macrophages can be both beneficial and detrimental after CNS injury. We previously showed rapid accumulation of macrophages in injured immature brain acutely after ischemia‐reperfusion. To determine whether these macrophages are microglia or invading monocytes, we subjected post‐natal day 7 (P7) rats to transient 3 h middle cerebral artery (MCA) occlusion and used flow cytometry at 24 and 48 h post‐reperfusion to distinguish invading monocytes (CD45high/CD11b+) from microglia (CD45low/medium/CD11b+). Inflammatory cytokines and chemokines were determined in plasma, injured and contralateral tissue 1–24 h post‐reperfusion using ELISA‐based cytokine multiplex assays. At 24 h, the number of CD45+/CD11b+ cells increased 3‐fold in injured compared to uninjured brain tissue and CD45 expression shifted from low to medium with less than 10% of the population expressing CD45high. MCA occlusion induced rapid and transient asynchronous increases in the pro‐inflammatory cytokine IL‐β and chemokines cytokine‐induced neutrophil chemoattractant protein 1 (CINC‐1) and monocyte‐chemoattractant protein 1 (MCP‐1), first in systemic circulation and then in injured brain. Double immunofluorescence with cell‐type specific markers showed that multiple cell types in the injured brain produce MCP‐1. Our findings show that despite profound increases in MCP‐1 in injured regions, monocyte infiltration is low and the majority of macrophages in acutely injured regions are microglia.

Carbon dioxide reactivity and pressure autoregulation of brain tissue oxygen.

OBJECTIVETo describe the normal relationships between brain tissue oxygen tension (PbrO2) and physiological parameters of systemic blood pressure and CO2 concentrations. METHODSLicox Clark-type oxygen probes (GMS mbH, Kiel, Germany) were inserted in the frontal white matter of 12 swine maintained under general anesthesia with a 1.0 fraction of inspired oxygen (FiO2). In seven swine, alterations in end-tidal carbon dioxide (ET-CO2) concentration (range, 13–72 mm Hg) were induced via hyperventilation or instillation of CO2 into the ventilation circuit. In nine swine, mean arterial pressure (MAP) (range, 33–200 mm Hg) was altered; phenylephrine was used to induce hypertension, and a nitroprusside-esmolol combination or systemic hemorrhage was used for hypotension. Quantitative cerebral blood flow (CBF) was measured in two animals by using a thermal diffusion probe. RESULTSMean baseline PbrO2 was 41.9 ± 11.3 mm Hg. PbrO2 varied linearly with changes in ET-CO2, ranging from 20 to 60 mm Hg (r2 = 0.70). The minimum PbrO2 with hypocarbia was 5.9 mm Hg, and the maximum PbrO2 with hypercarbia was 132.4 mm Hg. PbrO2 varied with MAP in a sigmoid fashion suggestive of pressure autoregulation between 60 and 150 mm Hg (r2 = 0.72). The minimum PbrO2 with hypotension was 1.4 mm Hg, and the maximum PbrO2 with hypertension was 97.2 mm Hg. In addition, CBF correlated linearly with PbrO2 during CO2 reactivity testing (r2 = 0.84). CONCLUSIONIn the uninjured brain, PbrO2 exhibits CO2 reactivity and pressure autoregulation. The relationship of PbrO2 with ET-CO2 and MAP appears to be similar to those historically established for CBF with ET-CO2 and MAP. This suggests that, under normal conditions, PbrO2 is strongly influenced by factors that regulate CBF.

Measurement of ventricular volumes in the dog by nuclear magnetic resonance imaging

Nuclear magnetic resonance (NMR) imaging is a new, noninvasive approach for imaging the cardiovascular system. Being a three-dimensional technique, NMR imaging has the capability of measuring volumes without the need for assumptions regarding ventricular geometry. In this study, the technique was validated in 19 excised dog hearts, filled with silicone-rubber, imaged using a multislice spin-echo sequence. The volume of the cavity in each slice was calculated from the number of pixels outlined for each slice multiplied by the pixel volume. Ventricular volumes measured by NMR imaging were highly correlated with cast volumes measured by water displacement: right ventricle (RV):RVNMR = 1.05 RVcast - 1.62; r = 0.99, SEE = 0.96 ml; left ventricle (LV):LVNMR = 0.98 LVcast + 0.35, r = 0.98, SEE = 1.48 ml. After validation in casts, NMR imaging volumes were measured in eight living dogs using a multiphasic gated technique to obtain images at 5, 105, 205, 305 and 405 ms after the QRS complex. Cardiac output (CO) and stroke volume (SV) measured by NMR imaging were significantly correlated with thermodilution (TD) measurements (CONMR = 0.63 COTD + 0.51 liters/min; r = 0.78, SEE = 0.57 liters/min; SVNMR = 0.67 SVTD + 1.95 ml; SEE = 5.58 ml). Right and left stroke volumes were closely related (LVSVNMR = 0.9 RVSVNMR + 1.75; r = 0.94, SEE = 4.32 ml), with the slope and intercept of the regression line showing no difference from 1 and 0, respectively. However, volumes determined by NMR imaging underestimated the thermodilution measurements, presumably reflecting the inability to obtain a true systolic image with the present sampling rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of Severity of Myocardial Injury on Distribution of Macromolecules: Extravascular Versus Intravascular Gadolinium-Based Magnetic Resonance Contrast Agents☆

OBJECTIVES This study sought to 1) compare the distribution of extravascular (573 Da) and intravascular (92 kDa) magnetic resonance (MR) contrast agents in reperfused infarcted myocardium, and 2) investigate the effect of injury severity on these distribution patterns. BACKGROUND Myocardial distribution of low and high molecular weight contrast agents depends on vascular permeability, diffusive/convective transport within the interstitium and accessibility of the intracellular compartment (cellular integrity). METHODS To vary the severity of myocardial injury, 72 rats were subjected to 20, 30, 45 or 75 min (n = 18, respectively) of coronary artery occlusion. After 2 h of reflow, the animals received either 0.05 mmol/kg of gadolinium-diethylenetriaminepentaacetic acid-bismethylamide (Gd-DTPA-BMA) (n = 24), (Gd-DTPA)30-albumin (n = 24) or saline (control group, n = 24). Three minutes after injection, the hearts were excised and imaged (spin-echo imaging parameters: repetition time 300 ms, echo time 8 ms, 2-tesla system), followed by triphenyltetrazolium chloride staining for infarct detection and sizing. RESULTS Histomorphometric and MR infarct size (expressed as percent of slice surface) correlated well: r = 0.96 for Gd-DTPA-BMA; r = 0.95 for (Gd-DTPA)30-albumin. On Gd-DTPA-BMA-enhanced images, reperfused myocardial infarctions were homogeneously enhanced. The ratio of signal intensity of infarcted/ normal myocardium increased with increasing duration of ischemia (overall p < 0.0001, analysis of variance [ANOVA]), indicating an increase in the distribution volume of Gd-DTPA-BMA in postischemic myocardium. On (Gd-DTPA)30-albumin-enhanced images, reperfused infarctions consisted of a bright border zone and a less enhanced central core. The extent of the core increased with increasing duration of ischemia (overall p value < 0.0001, ANOVA). CONCLUSIONS At 2 h of reperfusion, the distribution of MR contrast agents in postischemic myocardium is 1) specific for extravascular and intravascular agents, and 2) modulated by the duration of ischemia.

Magnetic resonance imaging of cardiac transplants: the evaluation of rejection of cardiac allografts with and without immunosuppression.

The purpose of this study was to evaluate the potential of magnetic resonance imaging (MRI) in vivo for the characterization of tissue changes associated with acute myocardial rejection after cardiac transplantation. Of 15 dogs that underwent heterotopic cardiac transplantation, six served as untreated controls, and nine received immunosuppressive therapy (25 mg/kg/day cyclosporine, 1 mg/kg/day prednisone). Serial electrocardiographically gated MRI (spin-echo technique) and histologic examinations of allograft biopsy samples were performed for each dog at 2 to 3, 7 to 10, 14 to 17, and 26 to 29 days after transplantation and immediately after animals were killed. Untreated allografts showed a significant increase (p less than .01) in T2 (spin-spin) relaxation time (T2 = 66 +/- 8 msec) and intensity values compared with values in the native hearts (T2 = 44 +/- 6 msec) as early as 1 week after transplantation. The significant difference in T2 values could be observed in vivo as well as on postmortem examination and corresponded to histologic progression of the rejection process. There was no significant difference in T1, T2, or intensity values in cyclosporine-treated allografts and native hearts except in two dogs in which T2 relaxation times and signal intensity in the transplanted hearts increased simultaneously with histologic evidence of rejection, indicating failure of immunosuppressive therapy. There was a significant correlation between histologic grading of severity of rejection and T2 relaxation times of the cardiac transplants (r = .72). Likewise, there was a significant linear relationship between T2 values in vivo and percent water content when the differences between native hearts and allografts were compared (r = .92, p less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Evolution of Brain Injury After Transient Middle Cerebral Artery Occlusion in Neonatal Rats

BACKGROUND AND PURPOSE Stroke in preterm and term babies is common and results in significant morbidity. The vulnerability and pathophysiological mechanisms of neonatal cerebral ischemia-reperfusion may differ from those in the mature cerebral nervous system because of the immaturity of many receptor systems and differences in metabolism in neonatal brain. This study details the neuropathological sequelae of reperfusion-induced brain injury after transient middle cerebral artery (MCA) occlusion in the postnatal day 7 (P7) rat. METHODS P7 rats were subjected to 3 hours of MCA occlusion followed by reperfusion or sham surgery. Diffusion-weighted MRI was performed during MCA occlusion, and maps of the apparent diffusion coefficient (ADC) were constructed. Contrast-enhanced MRI was performed in a subset of animals before and 20 minutes after reperfusion. Triphenyltetrazolium chloride (TTC) staining of the brain was performed 24 hours after reperfusion. Immunohistochemistry to identify astrocytes (glial fibrillary acidic protein), reactive microglia (ED-1), and neurons (microtubule-associated protein 2) and cresyl violet staining were done 4, 8, 24, and 72 hours after reperfusion. RESULTS On contrast-enhanced MRI, nearly complete disruption of cerebral blood flow was evident in the vascular territory of the MCA during occlusion. Partial restoration of blood flow occurred after removal of the suture. A significant decrease of the ADC, indicative of early cytotoxic edema, occurred in anatomic regions with a disrupted blood supply. The decline in ADC was associated with TTC- and cresyl violet-determined brain injury in these regions 24 hours later. The ischemic core was rapidly infiltrated with reactive microglia and was surrounded by reactive astroglia. CONCLUSIONS In P7 rats, transient MCA occlusion causes acute cytotoxic edema and severe unilateral brain injury. The presence of a prominent inflammatory response suggests that both the ischemic episode and the reperfusion contribute to the neuropathological outcome.