Kathryn G. Todd

Endothelial Progenitor Cells During Cerebrovascular Disease

Background and Purpose— Endothelial progenitor cells (EPCs) are associated with endothelial repair after ischemia in cardiac or peripheral circulation. There are no reports of EPCs with cerebrovascular disease. We present our experience with EPCs in patients with cerebrovascular disease. Summary of Report— EPC counts differed significantly (P<0.001) between stroke patients (acute stroke: median 4.75 and range 0 to 33; stable stroke: median 7.25 and range 0 to 43) and control subjects (median 15.5 and range 4.3 to 50), independent of age. The level of EPCs was significantly correlated with the Framingham coronary risk score (FCRS) (&rgr;=−0.349; P=0.002). Conclusions— Similar to cardiac experience, the low EPC levels may play a role in the pathophysiology of cerebrovascular disease.

Brain-derived neurotrophic factor and neurotrophin-3 activate striatal dopamine and serotonin metabolism and related behaviors: interactions with amphetamine

To investigate behavioral and neurochemical effects of neurotrophic factors in vivo, rats received continuous 14 d infusions of either brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or vehicle unilaterally into the substantia nigra. BDNF and NT-3 decreased body weights, an effect that was sustained over the infusion period. BDNF elevated daytime and nocturnal locomotion compared with infusions of vehicle or NT-3. At 2 weeks, a systemic injection of amphetamine (1.5 mg/kg, s.c.) increased the frequencies and durations of rotations contraversive to the side of BDNF and NT-3 infusions. Both factors attenuated amphetamine-induced locomotion without affecting amphetamine- induced stereotyped behaviors such as sniffing, head movements, and snout contact with cage surfaces. Only BDNF induced backward walking, and this response was augmented by amphetamine. BDNF, but not NT-3, increased dopamine turnover in the striatum ipsilateral to the infusion relative to the contralateral striatum. Both trophic factors decreased dopamine turnover in the infused substantia nigra relative to the contralateral hemisphere and increased 5-HT turnover in the striatum of both sides. Contraversive rotations were positively correlated with dopamine content decreases and 5-HT turnover increases in the striatum ipsilateral to the infused substantia nigra. Backward walking was positively correlated with increased dopamine and 5-HT turnover in the striatum of the infused hemisphere. Supranigral infusions of BDNF and NT-3 alter circadian rhythms, spontaneous motor activity, body weights, and amphetamine-induced behaviors including locomotion and contraversive rotations. These behavioral effects of the neurotrophins are consistent with a concomitant activation of dopamine and 5-HT systems in vivo.

Microglia in cerebral ischemia: molecular actions and interactions.

The precise role of microglia in stroke and cerebral ischemia has been the subject of debate for a number of years. Microglia are capable of synthesizing numerous soluble and membrane-bound biomolecules, some known to be neuroprotective, some neurotoxic, whereas others have less definitive bioactivities. The molecular mechanisms through which microglia activate these molecules have thus become an important area of ischemia research. Here we provide a survey review that summarizes the key actions of microglial factors in cerebral ischemia including complement proteins, chemokines, pro-inflammatory cytokines, neurotrophic factors, hormones, and proteinases, as well several important messenger molecules that play a part in how these factors respond to extracellular signals during ischemic injuries. We also provide some new perspectives on how microglial intracellular signaling may contribute to the seemingly contradictory roles of several microglial effector molecules.

Oxytocin in the central nervous system and sexual behaviour in male rats

Concentrations of oxytocin (OT) and vasopressin (AVP) were measured in cerebrospinal fluid (CSF) obtained from the cisterna magna of freely moving rats. Basal levels of OT and AVP were approximately 9 fmol/ml in both male and female. In the male rats this increased to approximately 18 fmol/ml 5 min after ejaculation, and 27 fmol/ml 20 min after ejaculation. No increase from basal levels occurred when males were placed with unreceptive females, or alone in the test environment. AVP levels were unchanged in any condition. Preliminary investigations indicate that discrete electrolytic lesions to the lateral and posterior parvocellular hypothalamic paraventricular nucleus (PVN) abolished this ejaculation-associated increase in CSF OT, prolonged mount and intromission latencies and reduced the absolute postejaculatory interval (PEI). We conclude that intracerebrally projecting OT systems may be activated during coitus and may contribute to the mechanisms underlying postejaculatory refractoriness.

Extracellular glutamate and other amino acids in experimental intracerebral hemorrhage: An in vivo microdialysis study

ObjectiveTo determine whether extracellular concentrations of glutamate and other amino acids are significantly elevated after intracerebral hemorrhage and, if so, the temporal characteristics of these changes. Although the role of excitotoxic amino acids, particularly that of glutamate, has been described in ischemic stroke and head trauma, no information exists regarding their possible contribution to the pathogenesis of neuronal injury in intracerebral hemorrhage. DesignProspective, controlled, laboratory trial. SettingsAnimal research laboratory. SubjectsSixteen anesthetized New Zealand rabbits. InterventionWe introduced intracerebral hemorrhage in each of eight anesthetized New Zealand rabbits by injecting 0.4 mL of autologous blood under arterial pressure into the deep gray matter of the cerebrum. Measurements and Main ResultsExtracellular fluid samples were collected from the perihematoma region and contralateral (right) hemisphere by in vivo microdialysis at 30-min intervals for 6 hrs. Corresponding samples were similarly collected from both hemispheres in each of eight control animals that underwent needle placement without introduction of a hematoma. Concentrations of amino acids (glutamate, aspartate, asparagine, glycine, taurine, and &ggr;-aminobutyric acid) in the samples were measured by use of high-pressure liquid chromatography with fluorescence detection. Glutamate concentrations (mean ± sem) were significantly higher in the hemisphere ipsilateral to the hematoma than in the contralateral hemisphere (92 ± 22 pg/&mgr;L vs. 22 ± 6 pg/&mgr;L) at 30 mins after hematoma creation. A significant increase was observed at 30 mins posthematoma creation in the hemisphere ipsilateral to the hematoma compared with the baseline value. A nonsignificant increase in glutamate concentration persisted in the hemisphere ipsilateral to the hematoma, ranging from 134% to 187% of baseline value between 1 and 5 hrs after hematoma creation. In the hemisphere ipsilateral to the hematoma, a three-fold increase in the concentration of glycine was observed at 30 mins after hematoma creation compared with the baseline level (890 ± 251 pg/&mgr;L vs. 291 ± 73 pg/&mgr;L). There was a significant difference between the hemisphere ipsilateral to the hematoma compared with the ipsilateral (corresponding) hemisphere of the control group at 30 mins posthematoma (890 ± 251 pg/&mgr;L vs. 248 ± 66 pg/&mgr;L). A similar transient increase was observed in taurine and asparagine concentrations at 30 mins after hematoma creation, compared with baseline measurements. Taurine concentrations in the hemisphere ipsilateral to the hematoma were significantly higher than the ipsilateral hemisphere of the control group (622 ± 180 pg/&mgr;L vs. 202 ± 64 pg/&mgr;L) at 30 mins after hematoma creation. ConclusionsThe present study suggests that glutamate and other amino acids accumulate transiently in extracellular fluids in the perihematoma region during the early period of intracerebral hemorrhage. The exact role of these amino acids in the pathogenesis of neuronal injury observed in intracerebral hemorrhage needs to be defined.

Differential regulation of trophic and proinflammatory microglial effectors is dependent on severity of neuronal injury.

Microglial activation has been reported to promote neurotoxicity and also neuroprotective effects. A possible contributor to this dichotomy of responses may be the degree to which proximal neurons are injured. The aim of this study was to determine whether varying the severity of neuronal injury influenced whether microglia were neuroprotective or neurotoxic. We exposed cortical neuronal cultures to varying degrees of hypoxia thereby generating mild (<20% death, 30min hypoxia), moderate (40–60% death, 2 h hypoxia), or severe (>70% death, 6 h hypoxia) injuries. Twenty‐four hours after hypoxia, the media from the neuronal cultures was collected and incubated with primary microglial cultures for 24 h. Results showed that the classic microglial proinflammatory mediators including inducible nitric oxide synthase, tumor necrosis factor α, and interleukin‐1‐β were upregulated only in response to mild neuronal injuries, while the trophic microglial effectors brain‐derived neurotrophic factor and glial cell line‐derived neurotrophic factor were upregulated in response to all degrees of neuronal injury. Microglia stimulated with media from damaged neurons were co‐cultured with hypoxic neurons. Microglia stimulated by moderate, but not mild or severe damage were neuroprotective in these co‐cultures. We also showed that the severity‐dependent phenomenon was not related to autocrine microglial signaling and was dependent on the neurotransmitters released by neurons after injury, namely glutamate and adenosine 5′‐triphosphate. Together our results show that severity of neuronal injury is an important factor in determining microglial release of “toxic” versus “protective” effectors and the resulting neurotoxicity versus neuroprotection.

Neuroprotection by memantine, a non-competitive NMDA receptor antagonist after traumatic brain injury in rats.

This study investigated whether memantine, a non-competitive NMDA receptor antagonist is neuroprotective after traumatic brain injury (TBI) induced in adult rats with a controlled cortical impact device. TBI led to significant neuronal death in the hippocampal CA2 and CA3 regions (by 50 and 59%, respectively), by 7 days after the injury. Treatment of rats with memantine (10 and 20 mg/Kg, i.p.) immediately after the injury significantly prevented the neuronal loss in both CA2 and CA3 regions. This is the first study showing the neuroprotective potential of memantine to prevent the TBI-induced neuronal damage.

Antenatal Sildenafil Treatment Attenuates Pulmonary Hypertension in Experimental Congenital Diaphragmatic Hernia

Background— Lung hypoplasia and persistent pulmonary hypertension of the newborn limit survival in congenital diaphragmatic hernia (CDH). Unlike other diseases resulting in persistent pulmonary hypertension of the newborn, infants with CDH are refractory to inhaled nitric oxide (NO). Nitric oxide mediates pulmonary vasodilatation at birth in part via cyclic GMP production. Phosphodiesterase type 5 (PDE5) limits the effects of NO by inactivation of cyclic GMP. Because of the limited success in postnatal management of CDH, we hypothesized that antenatal PDE5 inhibition would attenuate pulmonary artery remodeling in experimental nitrofen-induced CDH. Methods and Results— Nitrofen administered at embryonic day 9.5 to pregnant rats resulted in a 60% incidence of CDH in the offspring and recapitulated features seen in human CDH, including structural abnormalities (lung hypoplasia, decreased pulmonary vascular density, pulmonary artery remodeling, right ventricular hypertrophy), and functional abnormalities (decreased pulmonary artery relaxation in response to the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide). Antenatal sildenafil administered to the pregnant rat from embryonic day 11.5 to embryonic day 20.5 crossed the placenta, increased fetal lung cyclic GMP and decreased active PDE5 expression. Antenatal sildenafil improved lung structure, increased pulmonary vessel density, reduced right ventricular hypertrophy, and improved postnatal NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide–induced pulmonary artery relaxation. This was associated with increased lung endothelial NO synthase and vascular endothelial growth factor protein expression. Antenatal sildenafil had no adverse effect on retinal structure/function and brain development. Conclusions— Antenatal sildenafil improves pathological features of persistent pulmonary hypertension of the newborn in experimental CDH and does not alter the development of other PDE5-expressing organs. Given the high mortality/morbidity of CDH, the potential benefit of prenatal PDE5 inhibition in improving the outcome for infants with CDH warrants further studies.

Matrix metalloproteinase activation and blood–brain barrier breakdown following thrombolysis

Thrombolysis with tissue plasminogen activator (tPA) is the only pharmacotherapy available for cerebral ischemia. However, the use of tPA can increase the risk of hemorrhage due to blood-brain barrier (BBB) breakdown. Recent evidence suggests that increased activation of matrix metalloproteinases (MMPs) may be involved in this breakdown. This study examines the temporal profile of MMP-2 and -9 following tPA administration to ischemic rats. Male Sprague-Dawley rats were randomly assigned to one of four groups (Sham-tPA; Sham-Saline; Ischemia-tPA; Ischemia-Saline; group n = 6, total N = 120). Focal embolic ischemia was induced by middle cerebral artery occlusion through injection of an autologous clot. One hour post-surgery, tPA (10 mg/kg) or saline was delivered intravenously and animals were euthanized at 3, 6, 12, or 24 h after onset of ischemia. Infarct volume was measured by TTC staining; BBB components examined immunohistochemically; and MMP activation measured by gelatin zymography. Our results show that tPA significantly reduced infarct volumes (overall infarct volume-Sham-tPA: 5.80 +/- 4.55 [mean +/- SE]; Sham-Saline: 5.00 +/- 4.23; Ischemia-tPA: 186.1 +/- 73.45; Ischemia-Saline: 284.8 +/- 88.74; all P < 0.05). Treatment with tPA was also associated with the activation of MMP-9 at 6, 12, and 24 h following ischemia. No temporal changes were observed in MMP-2 activation, although tPA administration increased its activity compared to saline treatment. Analyses of immunohistochemistry showed that destruction of components of the BBB followed MMP-9 activation. Thus, increased MMP-9 activation may, in part, be responsible for the increases in hemorrhagic transformation reported with use of tPA. Our study is the first to demonstrate the temporal profile of MMP activation following thrombolysis with tPA in a model of thrombotic focal cerebral ischemia.

Laser Speckle Contrast Imaging of Collateral Blood Flow during Acute Ischemic Stroke

Collateral vasculature may provide an alternative route for blood flow to reach the ischemic tissue and partially maintain oxygen and nutrient support during ischemic stroke. However, much about the dynamics of stroke-induced collateralization remains unknown. In this study, we used laser speckle contrast imaging to map dynamic changes in collateral blood flow after middle cerebral artery occlusion in rats. We identified extensive anastomatic connections between the anterior and middle cerebral arteries that develop after vessel occlusion and persist for 24 hours. Augmenting blood flow through these persistent yet dynamic anastomatic connections may be an important but relatively unexplored avenue in stroke therapy.