Biomedical subjects

M Chopp

Publications and source records attributed to M Chopp.

At least 55 records · Page 3Linked to original sources

PubMed1999

Cerebral microvascular obstruction by fibrin is associated with upregulation of PAI-1 acutely after onset of focal embolic ischemia in rats.

The mechanisms underlying cerebral microvascular perfusion deficit resulting from occlusion of the middle cerebral artery (MCA) require elucidation. We, therefore, tested the hypothesis that intravascular fibrin deposition in situ directly obstructs cerebral microcirculation and that local changes in type 1 plasminogen activator inhibitor (PAI-1) gene expression contribute to intravascular fibrin deposition after embolic MCA occlusion. Using laser-scanning confocal microscopy (LSCM) in combination with immunofluorescent staining, we simultaneously measured in three dimensions the distribution of microvascular plasma perfusion deficit and fibrin(ogen) immunoreactivity in a rat model of focal cerebral embolic ischemia (n = 12). In addition, using in situ hybridization and immunostaining, we analyzed expression of PAI-1 in ischemic brain (n = 13). A significant (p < 0.05) reduction of cerebral microvascular plasma perfusion accompanied a significant (p < 0.05) increase of intravascular and extravascular fibrin deposition in the ischemic lesion. Microvascular plasma perfusion deficit and fibrin deposition expanded concomitantly from the subcortex to the cortex during 1 and 4 hr of embolic MCA occlusion. Three-dimensional analysis revealed that intravascular fibrin deposition directly blocks microvascular plasma perfusion. Vascular plugs contained erythrocytes, polymorphonuclear leukocytes, and platelets enmeshed in fibrin. In situ hybridization demonstrated induction of PAI-1 mRNA in vascular endothelial cells in the ischemic region at 1 hr of ischemia. PAI-1 mRNA significantly increased at 4 hr of ischemia. Immunohistochemical staining showed the same pattern of increased PAI-1 antigen in the endothelial cells. These data demonstrate, for the first time, that progressive intravascular fibrin deposition directly blocks cerebral microvascular plasma perfusion in the ischemic region during acute focal cerebral embolic ischemia, and upregulation of the PAI-1 gene in the ischemic lesion may foster fibrin deposition through suppression of fibrinolysis.

Animals↗

PubMed1999

Neuroprotective effects of progesterone after transient middle cerebral artery occlusion in rat.

Treatment of focal cerebral ischemia in the rat with intraperitoneal administration of progesterone dissolved in dimethyl sulfoxide (DMSO) has demonstrated therapeutic efficacy. In the present study we test whether iv administration of water soluble progesterone 2 h after the onset of middle cerebral artery occlusion provides therapeutic benefit for the treatment of stroke. In addition, we perform a battery of functional tests: rotarod, adhesive-backed somatosensory, and neurological score, as well as a dose-response study. The data indicate that iv administration of progesterone at a dose of 8 mg/kg significantly reduces the volume of cerebral infarction and significantly improves outcome on the array of functional measures employed. Treatment with 4 mg/kg or 32 mg/kg of progesterone failed to provide any therapeutic benefit. Progesterone, a non toxic, clinically employed, pluripotent therapeutic agent which targets both neuroprotective as well as neuroregenerative strategies, may have important therapeutic benefits for the treatment of stroke.

Animals↗

PubMed1999

Receptor tyrosine kinase tie 1 mRNA is upregulated on cerebral microvessels after embolic middle cerebral artery occlusion in rat.

Tie 1 is an endothelial specific transmembrane receptor tyrosine kinase and may be required during angiogenesis. Using in situ hybridization, we measured tie 1 mRNA in ischemic brain (n=15). Rats were subjected to middle cerebral artery (MCA) occlusion by a single fibrin rich clot. Expression of tie 1 was not detected in non ischemic brain. Cerebral microvessels expressed tie 1 in the ischemic lesion as early as 2 h after MCA occlusion. The number of microvessels containing tie 1 mRNA decreased in the ischemic lesion at 8 h after MCA occlusion. However, expression of tie 1 increased on microvessels at 24 h and 14 days after ischemia and tie 1 was primarily localized to the microvessels bordering pan necrotic tissue. Ninety-seven percent of cerebral vessels which expressed tie 1 mRNA had diameters of 3.7+/-0.17 microm. Our findings suggest a role for tie 1 in cerebral microvascular remodeling after embolic stroke.

Animals↗

PubMed1999

Progesterone is neuroprotective after acute experimental spinal cord trauma in rats.

STUDY DESIGN: A standardized rat contusion model was used to test the hypothesis that progesterone significantly improves neurologic recovery after a spinal cord injury that results in incomplete paraplegia. OBJECTIVES: To compare the effect of progesterone versus a variety of control agents to determine its effectiveness in promoting neurologic recovery after an incomplete rat spinal cord injury. SUMMARY OF BACKGROUND DATA: Progesterone is a neurosteroid, possessing a variety of functions in the central nervous system. Exogenous progesterone has been shown to improve neurologic function after focal cerebral ischemia and facilitates cognitive recovery after cortical contusion in rats. METHODS: A standardized rat contusion model of spinal cord injury using the New York University impactor that resulted in rats with incomplete paraplegia was used. Forty mature male Sprague-Dawley rats were randomly assigned to four groups: laminectomy with sham contusion, laminectomy with contusion without pharmacologic treatment, laminectomy with contusion treated with dimethylsulfoxide and dissolved progesterone, and laminectomy with contusion treated with dimethylsulfoxide. Functional status was assessed weekly using the Basso-Beattie-Bresnehan (BBB) locomotor rating scale for 6 weeks, after which the animals were killed for histologic studies. RESULTS: Rats treated with progesterone had better outcomes (P = 0.0017; P = 0.0172) with a BBB score of 15.5, compared with 10.0 in the dimethylsulfoxide control group and 12.0 in the spinal cord contusion without pharmacologic intervention group. This was corroborated in histologic analysis by relative sparing of white matter tissue at the epicenter of the injury in the progesterone-treated group (P < 0.05). CONCLUSIONS: Rats treated with progesterone had a better clinical and histologic outcome compared with the various control groups. These results indicate potential therapeutic properties of progesterone in the management of acute spinal cord injury.

Acute Disease↗

PubMed1999

Three-dimensional measurement of cerebral microvascular plasma perfusion, glial fibrillary acidic protein and microtubule associated protein-2 immunoreactivity after embolic stroke in rats: a double fluorescent labeled laser-scanning confocal microscopic study.

Early astroglial response to post-ischemic microvascular hypoperfusion may contribute to progressive cerebral microcirculatory impairment and ischemic neuronal injury. Using laser-scanning confocal microscopy and three fluorescent probes, we measured in three-dimensions cerebral microvascular plasma perfusion, astrocytic reactivity, and neuronal injury assessed by fluorescein isothiocyanate (FITC)-dextran, GFAP immunoreactivity, and microtubule associated protein-2 (MAP2) immunoreactivity, respectively, in rats subjected to 2 h of middle cerebral artery occlusion. Three-dimensional quantitative analysis revealed that 2 h of embolic ischemia resulted in a significant (P<0.05) reduction of cerebral microvascular plasma perfusion in the ipsilateral cortex and subcortex. Tissue within the ipsilateral cortex and subcortex with low plasma perfusion exhibited a significant (P<0.05) increase in GFAP immunoreactivity compared with the homologous contralateral tissue. Three-dimensional re-constructed images showed that prominent GFAP immunoreactive astrocytes surrounded large vessels with decreased plasma perfusion in downstream capillaries in the ipsilateral MCA territory when compared to the vessels in the contralateral homologous tissue. Triple fluorescence probe-stained sections showed that tissue with decreased plasma perfusion and with increased GFAP immunoreactivity was accompanied by a reduction of MAP2 immunoreactivity. The present study demonstrates that an impairment of microvascular perfusion induces an early increase in GFAP immunoreactivity, and reactive astrocytes may contribute to a further reduction of cerebral microvascular plasma perfusion. The three-dimensional quantitative imaging analysis used in the present study provides a means to investigate parenchymal cellular responses to changes of cerebral microvascular plasma perfusion after MCA occlusion.

Animals↗

PubMed1999

Expression of nestin after traumatic brain injury in rat brain.

We tested the hypothesis that traumatic brain injury upregulates expression of nestin, an embryonic cell intermediate filament protein. Brain from rats (n=24) subjected to controlled cortical impact injury and sham operated (n=3) and normal (n=3) rats were processed for dual label immunohistochemical study to identify cellular expression of nestin. Our results show that in normal noninjured animals, nestin is expressed slightly and localized only in a few endothelial and subventricular cells. In contrast, at 1-4 weeks postinjury nestin is strongly expressed in astrocytes and microglia. The expression of nestin in astrocytes and microglia after traumatic brain injury support the hypothesis that injured cerebral tissue expresses developmental proteins, and that these proteins may promote recovery after injury.

Animals↗

PubMed1999

Temporal profile of nestin expression after focal cerebral ischemia in adult rat.

Nestin is an intermediate filament protein, transiently and abundantly expressed early in embryogenesis, e.g., in neuroepithelial cells, radial glia, germinal matrix cells and vascular cells. In the adult rat brain, nestin is only present in endothelial and select subventricular cells. We tested the hypothesis that after an experimental stroke, nestin expression is induced in glial cells and neurons. We measured the temporal profile of nestin expression after induction of focal cerebral ischemia in adult rats. Brain from rats (n=24) subjected to 2 h of transient middle cerebral artery occlusion (MCAo) and 3 h, 6 h, 12 h, 1 day, 2 days, 3 days, 7 days and 28 days (n=3, per time point) of reperfusion, and control sham operated (n=3) rats were processed for Western blotting to quantify nestin. Another set of brains from rats (n=28), subjected to 2 h of MCAo and 6 h, 12 h, 2 days, 7 days, 14 days, 21 days, and 28 days (n=4, per time point, except n=8 at 2 days) of reperfusion, and control sham operated (n=3) and normal (n=2) rats were processed by single and double labeled immunohistochemistry for cellular identification of nestin expression. By Western blotting, nestin within ischemic tissue increased slightly as early as 6 h, peaked at 7 days, and expression persisted for at least 4 weeks after 2 h of MCAo. By immunohistochemistry, nestin was expressed in astrocytes in the ischemic core from 6 to 12 h after MCAo. Nestin immunoreactivity was present in large numbers of astrocytes, and in scattered oligodendroglia and monocytes/macrophages in both the inner and outer boundary zones to the ischemic core at 1-7 days after MCAo. Nestin expression in glial cells declined at longer durations of survival, although for least 4 weeks after MCAo the nestin immunoreactivity delineated the boundary zone adjacent to the ischemic core. Nestin expression was present in some neurons localized to the outer boundary zone of the ischemic lesion in the cortex and striatum, and in most ependymal cells in the ventricular and subventricular zone (VZ/SVZ) from day 2 after MCAo and onward. The expression of nestin increased throughout the microvasculature in both the ischemic core and the boundary zone in all ischemic rats after 12 h of reperfusion. After stroke, nestin immunoreactivity in glial, neuronal and ependymal cells is suggestive of a protein expression pattern found in developing brain.

Animals↗

PubMed1999

Identification of cerebral ischemic lesions in rat using Eigenimage filtered magnetic resonance imaging.

An accurate noninvasive time-independent identification of an ischemic cerebral lesion is an important objective of magnetic resonance imaging (MRI). This study describes a novel application of a multiparameter MRI analysis algorithm, the Eigenimage (EI) filter, to experimental stroke. The EI is a linear filter that maximizes the projection of a desired tissue (ischemic tissue) while it minimizes the projection of undesired tissues (nonischemic tissue) onto a composite image called an eigenimage. Rats (n=26) were subjected to permanent middle cerebral artery occlusion. T2- and T1-weighted coronal MRI were acquired on separate groups of animals. The animals were immediately sacrificed after each imaging session for histopathological analysis of tissue at 4-8 h, 16-24 h, and 48-168 h after stroke onset. Lesion areas from MRI were defined using EI. The EI defined lesion areas were coregistered and warped to the corresponding histopathological sections. The ischemic lesion as defined by EI exhibited ischemic cell damage ranging from scattered acute cell damage to pan necrosis. Ischemic cellular damage was not detected in homologous contralateral hemisphere regions. EI lesion areas overlaid on histopathological sections were significantly correlated (r=0.92, p<0.05) acutely, (r=0.98, p<0.05) subacutely, and (r=0.99, p<0.05) chronically. These data indicate that EI methodology can accurately segment ischemic damage after MCA occlusion from 4-168 h after stroke.

Algorithms↗

PubMed1999

Endogenous plasminogen activator expression after embolic focal cerebral ischemia in mice.

Urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) play important roles in fibrinolysis, cell migration, tissue destruction, angiogenesis and tissue remodeling. u-PA and t-PA activity in tissue are tightly regulated by plasminogen activator inhibitor-1 (PAI-1). However, little is known of the activity of endogenous plasminogen activators (PAs) and PAI-1 in ischemic brain. To evaluate whether cerebral ischemic injury induces endogenous PAs and PAI-1, we measured PA activity from brain homogenates, and examined the expression of t-PA mRNA, u-PA mRNA and PAI-1 mRNA from brain homogenates in C57BL/6J mice (n=45) weighing 29-35 g in which the middle cerebral artery (MCA) was occluded by a fibrin-rich clot. Brain homogenates were prepared for direct casein zymography from control non-ischemic mice (n=4) and mice at 2 h (n=5), 4 h (n=5), and 24 h (n=4) after MCA occlusion (MCAO). Also, u-PA and t-PA knockout mice at 4 h (n=2, each) after MCAO were used as a negative control for direct casein zymography. Frozen sections for in situ zymography were obtained from control mice (n=2) and mice at 2 h, 4 h, and 24 h (n=2, per time point) after clot occlusion. Brain homogenates were prepared for reverse transcriptase-polymerase chain reaction (RT-PCR) to examine t-PA mRNA, u-PA mRNA and PAI-1 mRNA expression from control non-ischemic mice (n=4) and mice at 2 h (n=5), 4 h (n=5), and 24 h (n=5) after MCAO. By direct casein zymography, u-PA activity increased at 4 h (P<0.05), and 24 h (P<0.05) after stroke in the ischemic hemisphere compared with the non-ischemic mice. Activity of t-PA in ischemic brain was not significantly different from the control group. As measured by in situ zymography, PA activity, most likely u-PA, was present in the ischemic hemisphere. By RT-PCR, expression of PAI-1 mRNA, but not u-PA mRNA and t-PA mRNA, increased 3-, 15- and 25-folds in the ischemic hemisphere at 2 h, 4 h and 24 h after stroke, respectively, compared with control mice. This study demonstrates that PAI-1 mRNA and u-PA activity increase in mouse brain after stroke.

Animals↗

PubMed1999

The effect of age on expression of endogenous plasminogen activators after focal cerebral ischemia in mice.

We measured urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) activity in the brain of 2-3 month old and 6-8 month old mice subjected to 4 h of middle cerebral artery (MCA) occlusion. t-PA activity was present in all non-ischemic and ischemic young mouse brain. In contrast, t-PA activity was present in 46.7% of non-ischemic middle aged mouse brain and in 44.4% of ischemic middle aged mouse brain. u-PA activity was present in all young and middle aged non-ischemic brains.

Aging↗

PubMed1999

Increase in apoptosis and concomitant reduction of ischemic lesion volume and evidence for synaptogenesis after transient focal cerebral ischemia in rat treated with staurosporine.

We sought to determine whether induction of apoptosis alters an ischemic lesion. Rats were subjected to 2 h of middle cerebral artery occlusion (MCAo) and treated with staurosporine (n=8) or vehicle (n=4). Our data demonstrate that at 22 h after MCAo, staurosporine triggered a significant increase in apoptosis, a reduction of lesion volume and an increase of synaptophysin immunoreactivity, as compared to the control group.

Animals↗

PubMed1999

Apoptosis and expression of p53 response proteins and cyclin D1 after cortical impact in rat brain.

We measured the temporal profile and cellular identification of apoptosis in rat brain after cortical contusion injury. Double staining immunohistochemistry was also used to investigate the relationship between apoptotic cell death and selective protein expression associated with DNA damage and repair (p53, Bax, MDM2, WAF1, Gadd45, PCNA) and cell cycle protein, Cyclin D1, in male Wistar rats 48 h after injury. Cortical contusion was induced in male Wistar rats with a pneumatic impactor device. The animals were sacrificed at different times after trauma (1, 2, and 14 h and 1, 2, 4, 7 and 14 days; n=4 per time point). Sham-operated rats (n=4) and normal rats not subjected to any surgical procedure (n=4) were used as controls for temporal profile determination. Additional 11 rats were used for study of protein expression. Coronal brain sections were analyzed using an in situ terminal deoxynucleotdyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL), hematoxylin, and immunohistochemical double staining methods. Apoptotic cells were observed as early as 2 h after the impact. Apoptotic cell death peaked at 2 days, gradually tapering off afterward, although scattered apoptotic cells were detected at 2 weeks after the impact. The number of apoptotic cells at 2 days far exceeded their number at other times (p=0.009). Apoptotic cells were observed primarily in the cortex adjacent to the site of injury. In addition, apoptotic cells in conjunction with few injured cells were present in the ipsilateral hippocampus and localized to the granule layer of dentate gyrus. Our data indicate that DNA fragmentation is present in nearly all neurons subacutely after cortical contusion and persists for at least 2 weeks thereafter. Apoptosis is also present in neurons localized to the hilus of the dentate gyrus at a site remote from the area of injury suggesting a selective role for apoptosis in promoting secondary brain damage and dysfunction after traumatic brain injury. Using double staining, we were able to show that a great majority of apoptotic cells (>95%) were neurons and the rest were astrocytes and endothelial cells. Proteins associated with DNA damage and repair (p53, Bax, MDM2, WAF1, Gadd 45, PCNA) were expressed in the cytoplasm of normal cells of naive and sham rats. These proteins were translocated to the nuclei of apoptotic and injured cells at 48 h after cortical contusion. Cyclin D1 was not present in apoptotic cells. The differential expression of proteins associated with DNA damage, repair and the cell cycle protein Cyclin D1 in the contused brain suggest a potential role for these proteins in cell survival and apoptosis after cortical contusion.

Animals↗

PubMed1999

Increased therapeutic efficacy with rt-PA and anti-CD18 antibody treatment of stroke in the rat.

OBJECTIVE: To examine the efficacy of an antileukocyte adhesion antibody (anti-CD18) as an adjuvant for delayed (2 hours and 4 hours) thrombolytic therapy (recombinant human tissue plasminogen activator [rt-PA]) in middle cerebral artery occlusion (MCAO) in rats. BACKGROUND: Thrombolytic therapy with rt-PA is limited in its application by a short therapeutic window. METHODS: Male Wistar rats were subjected to MCAO by a single fibrin-rich clot. The rats were assigned to the following experimental groups: Experiment 1 (treatment 2 hours after embolization), 1) rt-PA, 2) anti-CD18 antibody, 3) rt-PA and anti-CD18 antibody, 4) immunoglobulin (Ig) G, and 5) vehicle; Experiment 2 (treatment 4 hours after occlusion), 1) rt-PA alone, 2) rt-PA and anti-CD18 antibody, and 3) nontreated control group. Neurologic deficits, infarction volume, hemorrhage, and brain myeloperoxidase (MPO) immunoreactivity were measured. RESULTS: Administration of rt-PA and anti-CD18 antibody 2 hours later reduced significantly (p < 0.05) the infarct volume and improved neurologic deficits compared with the vehicle-treated group. Treatment with rt-PA alone improved neurologic deficits significantly and reduced mean infarct volume compared with the vehicle-treated group. However, treatment with anti-CD18 antibody neither reduced infarct volume nor improved neurologic deficits compared with the IgG-treated group. The combination of rt-PA and anti-CD18 antibody treatment at 4 hours reduced significantly the infarct volume and MPO immunoreactive cells compared with rt-PA treatment alone at 4 hours, and reduced neurologic deficits compared with rt-PA treatment alone and compared with the nontreated animals. CONCLUSIONS: The combination of antileukocyte adhesion antibody and thrombolytic therapy may increase the therapeutic window for the treatment of stroke.

Adjuvants, Immunologic↗

PubMed1999

T1 and magnetization transfer at 7 Tesla in acute ischemic infarct in the rat.

T1 and magnetization transfer at a field strength of 7 Tesla were used to discriminate between water accumulation and protein mobilization in tissue undergoing infarction. Twelve rats subjected to acute stroke via intralumenal suture occlusion of the middle cerebral artery, and 19 controls, were studied. In MRI studies to 6 hr post-ictus, serial data acquisition allowed the measurement of cerebral blood flow (CBF), apparent diffusion coefficient of water (ADCw), equilibrium magnetization (M0) and T1, and equilibrium magnetization and T1 under an off-resonance partial saturation of the macromolecular pool (Msat and T1sat). Using these parameters, the apparent forward transfer rate of magnetization between the free water proton pool and the macromolecular proton pool, k(fa), was calculated. Regions of interest (ROIs) were chosen using depressed areas in maps of the ADCw. T1 measurements in bovine serum albumin at 7T were not affected by the mobility of the macromolecular pool (P > 0.2), but magnetization transfer between free water and protein depended strongly on the mobility of the macromolecular pool (P < 0.001). For 6 hr after ictus, k(fa) uniformly and strongly decreased in the region of the infarct (P < 0.0001). Ratios (ischemic/non-ischemic) of parameters M0, Msat, T1, and T1sat all uniformly and strongly increased in the infarct. The ratio T1/T1sat in the region of infarction showed that a progressive accumulation of free water in the region of interest was the major (>80%) contribution to the decrease in k(fa). There also existed a small contribution due to changes at the water-macromolecular interface, possibly due to proteolysis (P = 0.005).

Acute Disease↗

PubMed1999

The clot thickens--thrombolysis and combination therapies.

Stroke in the human is most frequently caused by an intra-arterial clot. In order to investigate human stroke, appropriate and relevant animal models must be selected. Since the only approved treatment of stroke is that of recombinant tissue plasminogen activator (rtPA) the models selected should be amenable to thrombolytic treatment. We therefore describe a new model of embolic stroke in the rat in which a fibrin rich clot is placed via the internal carotid artery (ICA) at the origin of the middle cerebral artery (MCA). Data are summarized describing treatment of embolic stroke with rtPA administered at various times after stroke, the use of combination antiadhesion molecule and rtPA therapy, and the application of MRI to monitor the temporal evolution of physiological changes within ischemic tissue with and without rtPA intervention and to predict therapeutic efficacy.

Animals↗

PubMed1999

High resolution quantitation of microvascular plasma perfusion in non-ischemic and ischemic rat brain by laser-scanning confocal microscopy.

Laser-scanning confocal microscopy (LSCM) was used to measure at high resolution cerebral plasma volumes (perfusion) using two fluorescent plasma markers in a rat model of embolic stroke. This application of LSCM to study the microvascular circulation in embolic stroke was developed as an alternative to autoradiography to measure cerebral perfusion. An additional benefit of LSCM is that it quantitates with great accuracy the structural relationships of the microcirculation to cells and the pathological alterations of the ischemic brain. Autoradiography allows only a quantitative analysis of cerebral perfusion. For example, in order to study the microcirculation and its relationship to blood brain barrier damage, the volume of perfused cerebral capillaries was measured by administering two fluorescent plasma markers (FITC-dextran and Evans blue) intravenously to a rat. Evans blue was administered before cerebral ischemia and FITC-dextran administered post-ischemia 1 min before sacrifice. Volumes of plasma perfusion were analyzed by means of a system developed for 3D analysis of fixed and stained serial brain histologies. Plasma volumes for the non-ischemic cerebral cortex were 1.00%+/-0.38% while plasma volumes in the caudate/putamen were 0.69%+/-0.17% in good agreement with the previously published values using the autoradiography method. The architecture of the capillaries in the ischemic core showed perfusion of Evans blue but there was no flow of FITC dextran. Our work represents a novel application of this technology to investigation of cerebral vascular disease and identifies its potential to become an important tool for investigation of cerebral pathology.

Animals↗

PubMed1999

Expression of cell cycle proteins (cyclin D1 and cdk4) after controlled cortical impact in rat brain.

We measured the expression of Cyclin D1 and its kinase cdk4, 48 h after induction of cortical contusion in the rat. Brain from rats (n = 6) subjected to controlled cortical impact injury and sham-operated (n = 3) and normal (n = 2) rats were processed for dual label immunohistochemical study to identify cellular expression of these cell cycle proteins. Antibodies against neurofilaments 68 and 200 and glial fibrillary acidic protein were employed to identify neurons and astrocytes, respectively, whereas microglia were identified using histochemical detection of IB4-isolectin. Double staining for DNA fragmentation detection, using terminal deoxynucleotdyl transferase mediated biotinylated deoxyuridine triphosphate nick end 3 'OH labeling (TUNEL) and antibodies for expression of Cyclin D1 and cdk4 was also performed. Cyclin D1 and cdk4 were selectively expressed in morphologically intact or injured neurons throughout the rat brain. Apoptotic cells were not immunoreactive to Cyclin D1 and cdk4. The selective expression of cell cycle proteins observed in nonapoptotic postmitotic neurons suggests a role for these proteins in the survival of cells after cortical contusion.

Animals↗

PubMed1999

Quantitation of microvascular plasma perfusion and neuronal microtubule-associated protein in ischemic mouse brain by laser-scanning confocal microscopy.

In an exposition of the technique of calculating distribution volumes from laser-scanning confocal microscopic (LSCM) data, three-dimensional images of the distribution of one or two fluorescent markers in mouse brain specimens were generated by LSCM and processed by a system developed for morphometric analysis of fixed and stained serial brain histologic samples. To determine the volume of perfused cerebral capillaries, one of two fluorescent plasma markers, either fluorescein isothiocyanate (FITC)-dextran or Evans blue, was intravenously administered to mice subjected to 1 hour of embolic middle cerebral artery (MCA) occlusion (n = 9) and to mice that were not operated on (n = 3); after 1 minute of circulation, brains were removed, immersion-fixed, and processed for LSCM. In some of these animals (n = 5), the volume of endogenous microtubule-associated protein-2 (MAP2) fluorescence was also determined using immunohistochemical staining. For mice that were not operated on, this methodology yielded highly localized volumes of (1) microvascular plasma, which agree with those determined for rodents by other techniques, and (2) MAP2 expression, which appears physiologically and morphologically reasonable. After 1 hour of MCA occlusion, the MAP2 volumes of distribution were less than 10% of normal in the ipsilateral hemisphere in which plasma perfusion essentially ceased. In conclusion, precise colocalization and quantitation of early ischemic neuronal damage and cerebral plasma perfusion deficit can be done with this three-dimensional, microphysiologic and microanatomic methodology.

Animals↗