Ilknur Ay

Potential Usefulness of Basic Fibroblast Growth Factor as a Treatment for Stroke

Within the past few years, a growing body of evidence has accumulated indicating that exogenously administered neurotrophic growth factors may limit the extent of acute ischemic neural injury and enhance functional neurorecovery following stroke. One of the most widely studied growth factor in this regard is basic fibroblast growth factor (bFGF). In preclinical studies, bFGF administered intravenously within hours after the onset of ischemia reduces infarct size, presumably due to direct protection of cells at the borders (penumbra) of cerebral infarction. On the other hand, if bFGF is administered intracisternally starting at one day after ischemia, infarct size is not reduced, but recovery of sensorimotor function of the impaired limbs is increased, presumably due to enhancement of new neuronal sprouting and synapse formation in the intact uninjured brain. Clinical trials of the intravenous administration of bFGF as a cytoprotective agent in acute stroke are in progress. Trials of the delayed administration of bFGF as a recovery-promoting agent in subacute stroke are anticipated.

Bimodal Thrombus Imaging: Simultaneous PET/MR Imaging with a Fibrin-targeted Dual PET/MR Probe—Feasibility Study in Rat Model

PURPOSE To image thrombus by using magnetic resonance (MR) imaging and positron emission tomography (PET) simultaneously in a rat arterial thrombus model with a dual PET/MR probe. MATERIALS AND METHODS Animal studies were approved by the institutional animal use committee. A dual PET/MR probe was synthesized by means of partial exchange of gadolinium for copper 64 ((64)Cu) in the fibrin-targeted MR probe EP-2104R. A preformed 25-mm thrombus was injected into the right internal carotid artery of a rat. Imaging was performed with a clinical 3.0-T MR imager with an MR-compatible human PET imager. Rats (n = 5) were imaged prior to and after systemic administration of the dual probe by using simultaneous PET/MR. The organ distribution of (64)Cu and gadolinium was determined ex vivo (n = 8), 2 hours after injection by using well counting and inductively coupled plasma mass spectrometry, respectively. Signal intensity ratios (SIRs) between the thrombus-containing and contralateral vessel were computed from PET images and MR data before and after probe administration. RESULTS The dual probe was synthesized with greater than 98% radiochemical purity. Thrombus enhancement was observed in all five animals at both MR (SIR([postprobe])/SIR([preprobe]) = 1.71 ± 0.35, P = .0053) and PET (SIR = 1.85 ± 0.48, P = .0087) after injection of the dual PET/MR probe. Ex vivo analysis at 2 hours after injection showed the highest (64)Cu and gadolinium concentrations, after the excretory organs (kidney and liver), to be in the thrombus. CONCLUSION A fibrin-targeted dual PET/MR probe enables simultaneous, direct MR and PET imaging of thrombus.

A Manganese Alternative to Gadolinium for MRI Contrast

Contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI) are routinely used to diagnose soft tissue and vascular abnormalities. However, safety concerns limit the use of iodinated and gadolinium (Gd)-based CT and MRI contrast media in renally compromised patients. With an estimated 14% of the US population suffering from chronic kidney disease (CKD), contrast media compatible with renal impairment is sorely needed. We present the new manganese(II) complex [Mn(PyC3A)(H2O)](-) as a Gd alternative. [Mn(PyC3A)(H2O)](-) is among the most stable Mn(II) complexes at pH 7.4 (log KML = 11.40). In the presence of 25 mol equiv of Zn at pH 6.0, 37 °C, [Mn(PyC3A)(H2O)](-) is 20-fold more resistant to dissociation than [Gd(DTPA)(H2O)](2-). Relaxivity of [Mn(PyC3A)(H2O)](-) in blood plasma is comparable to commercial Gd contrast agents. Biodistribution analysis confirms that [Mn(PyC3A)(H2O)](-) clears via a mixed renal/hepatobiliary pathway with >99% elimination by 24 h. [Mn(PyC3A)(H2O)](-) was modified to form a bifunctional chelator and 4 chelates were conjugated to a fibrin-specific peptide to give Mn-FBP. Mn-FBP binds the soluble fibrin fragment DD(E) with Kd = 110 nM. Per Mn relaxivity of Mn-FBP is 4-fold greater than [Mn(PyC3A)(H2O)](-) and increases 60% in the presence of fibrin, consistent with binding. Mn-FBP provided equivalent thrombus enhancement to the state of the art Gd analogue, EP-2104R, in a rat model of arterial thrombosis. Mn metabolite analysis reveals no evidence of dechelation and the probe was >99% eliminated after 24 h. [Mn(PyC3A)(H2O)](-) is a lead development candidate for an imaging probe that is compatible with renally compromised patients.

Vagus nerve stimulation reduces infarct size in rat focal cerebral ischemia.

BACKGROUND AND PURPOSE We sought to determine the effect of vagus nerve stimulation (VNS) on infarct size after transient focal cerebral ischemia in rats. METHODS Ischemia was produced by transient filament occlusion of the right middle cerebral artery. Stimulating electrodes were implanted on the cervical part of the right vagus nerve. Electrical stimulation was initiated 30 min after the induction of ischemia, and delivered for 30s at every 30 min for 3h in experimental group 1 and at every 5 min for 1h in experimental group 2. All the procedures were duplicated but no stimulus was delivered in the control group. Functional deficit was evaluated and animals were killed to determine the infarct size 24h after ischemia. RESULTS Ischemic lesion volume was smaller in VNS-treated animals as compared with control animals; the relative percentage of contralateral hemispheric volume that underwent infarction was 16.2+/-3.2% in the VNS and 33.0+/-5.0% in the control arms in experimental group 1 (p<0.05). The respective values for experimental group 2 were 19.8+/-0.5% and 37.9+/-2.6% (p<0.05). VNS-treated animals were significantly more likely to have better functional scores at 24h as compared with control animals. The functional score improved by 50% in experimental group 1 and 44% in experimental group 2 (p<0.05 for both groups). CONCLUSION VNS appears to offer protection against acute ischemic brain injury.

A glial cell line-derived neurotrophic factor (GDNF):tetanus toxin fragment C protein conjugate improves delivery of GDNF to spinal cord motor neurons in mice

Glial cell line-derived neurotrophic factor (GDNF) has shown robust neuroprotective and neuroreparative activities in various animal models of Parkinsons Disease or amyotrophic lateral sclerosis (ALS). The successful use of GDNF as a therapeutic in humans, however, appears to have been hindered by its poor bioavailability to target neurons in the central nervous system (CNS). To improve delivery of exogenous GDNF protein to CNS motor neurons, we employed chemical conjugation techniques to link recombinant human GDNF to the neuronal binding fragment of tetanus toxin (tetanus toxin fragment C, or TTC). The predominant species present in the purified conjugate sample, GDNF:TTC, had a molecular weight of approximately 80 kDa as determined by non-reducing SDS-PAGE. Like GDNF, addition of GDNF:TTC to culture media of neuroblastoma cells expressing GFRalpha-1/c-RET produced a dose-dependent increase in cellular phospho-c-RET levels. Treatment of cultured midbrain dopaminergic neurons with either GDNF or the conjugate similarly promoted both DA neuron survival and neurite outgrowth. However, in contrast to mice treated with GDNF by intramuscular injection, mice receiving GDNF:TTC revealed intense GDNF immunostaining associated with spinal cord motor neurons in fixed tissue sections. That GDNF:TTC provided neuroprotection of axotomized motor neurons in neonatal rats further revealed that the conjugate retained its GDNF activity in vivo. These results indicate that TTC can serve as a non-viral vehicle to substantially improve the delivery of functionally active growth factors to motor neurons in the mammalian CNS.

VEGF increases blood-brain barrier permeability to Evans blue dye and tetanus toxin fragment C but not adeno-associated virus in ALS mice

Entry of most compounds into the CNS is impeded by the blood-brain barrier (BBB). Because vascular endothelial growth factor (VEGF) is important in the formation and maintenance of the BBB and is known to modulate BBB permeability in newborn rodents, we tested the hypothesis that VEGF may enhance BBB permeability in adult mice. We examined the effect of VEGF on the CNS distribution of three different agents: a small molecule (Evans blue dye) that is known to bind plasma proteins, an exogenous protein (tetanus toxin fragment C; TTC), and a viral vector (recombinant adeno-associated virus serotype 2/5 marked with lacZ; rAAV2/5-lacZ). Pretreatment with VEGF (20 mug; i.v.) increased permeability of the BBB to Evans blue dye and TTC as detected by augmented concentrations of these substances in the cerebrum, brainstem, and spinal cord. By contrast, VEGF did not alter BBB permeability to AAV2/5-lacZ, as defined by beta-galactosidase activity assay. These data demonstrate the potential utility of VEGF for pharmacological modulation of the BBB, and indicate that the increase in BBB permeability mediated by VEGF is limited by the size of the delivered substance.

Vagus nerve stimulation inhibits cortical spreading depression.

Abstract Vagus nerve stimulation has recently been reported to improve symptoms of migraine. Cortical spreading depression is the electrophysiological event underlying migraine aura and is a trigger for headache. We tested whether vagus nerve stimulation inhibits cortical spreading depression to explain its antimigraine effect. Unilateral vagus nerve stimulation was delivered either noninvasively through the skin or directly by electrodes placed around the nerve. Systemic physiology was monitored throughout the study. Both noninvasive transcutaneous and invasive direct vagus nerve stimulations significantly suppressed spreading depression susceptibility in the occipital cortex in rats. The electrical stimulation threshold to evoke a spreading depression was elevated by more than 2-fold, the frequency of spreading depressions during continuous topical 1 M KCl was reduced by ∼40%, and propagation speed of spreading depression was reduced by ∼15%. This effect developed within 30 minutes after vagus nerve stimulation and persisted for more than 3 hours. Noninvasive transcutaneous vagus nerve stimulation was as efficacious as direct invasive vagus nerve stimulation, and the efficacy did not differ between the ipsilateral and contralateral hemispheres. Our findings provide a potential mechanism by which vagus nerve stimulation may be efficacious in migraine and suggest that susceptibility to spreading depression is a suitable platform to optimize its efficacy.

Molecular MRI of Intracranial Thrombus in a Rat Ischemic Stroke Model

Background and Purpose— Intracranial thrombus is a principal feature in most ischemic stroke, and thrombus location and size may correlate with outcome and response to thrombolytic therapy. EP-2104R is a fibrin-specific molecular MR agent that has previously been shown to enhance extracranial and venous sinus thrombi in animal models and, recently, in clinical trials. In this study, we examined whether this fibrin-specific MR probe could noninvasively characterize intracranial arterial thrombi. Methods— Embolic stroke was induced in adult rats by occlusion of the right internal carotid artery with an aged thrombus. We used diffusion-weighted imaging, time of flight angiography, and high-resolution 3-dimensional T1-weighted MRI at 4.7 T before and after use of contrast agents EP-2104R (n=6) and gadopentetate dimeglumine (n=5). Results— In all animals, MR angiography revealed a flow deficit and diffusion-weighted imaging showed hyperintensity consistent with ischemia. Using EP-2104R-enhanced MRI, we saw occlusive thrombi and vessel wall enhancement in all 6 animals with high contrast to noise relative to blood, whereas gadopentetate dimeglumine-injected animals showed no occlusive thrombus or vessel wall enhancement. The concentration of gadolinium in the thrombus after EP-2104R was 18 times that in the blood pool. Conclusions— EP-2104R-enhanced MRI successfully identifies intracranial thrombus in a rat embolic stroke model.

Vagus nerve stimulation reduces infarct size in rat focal cerebral ischemia: An unlikely role for cerebral blood flow

We sought to investigate the effect of cervical vagus nerve stimulation (VNS) on cerebral blood flow (CBF), infarct volume, and clinical outcome in a model of middle cerebral artery occlusion in rats. Electrical stimulation of the right and left vagus nerves was initiated 30min after the induction of the right-sided ischemia and lasted for 1h. Infarct size measurement revealed that the volume of ischemic damage was 41-45% smaller in animals receiving stimulation as compared with control animals. Both the right and left VNS caused subtle reduction in CBF during each 30-s stimulation period that quickly returned back to the baseline level at the end of each stimulation cycle. There was no significant effect of VNS on CBF during the entire 1-h stimulation period. The effect of VNS on tissue outcome was associated with better neurological outcome at both 1- and 3-day time points after the induction of ischemia. These findings suggest that VNS-induced protection against acute ischemic brain injury is not primarily mediated by changes in CBF, stimulation of both the right and left nerve have comparable effects, and VNS is effective after ipsilateral and contralateral focal ischemia.

Fibrin-targeted PET probes for the detection of thrombi.

There is an ongoing effort to develop better methods for noninvasive detection and characterization of thrombi. Here we describe the synthesis and evaluation of three new fibrin-targeted positron emission tomography (PET) probes (FBP1, FBP2, FBP3). Three fibrin-specific peptides were conjugated as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-monoamides at the C- and N-termini and chelated with (64)CuCl2. Probes were prepared with a specific activity ranging from 10 to 130 μCi/nmol. Both the peptides and the probes exhibited nanomolar dissociation constants (Kd) for the soluble fibrin fragment DD(E), although the Cu-DOTA derivatization resulted in a 2-3 fold loss in affinity relative to the parent peptide. Biodistribution and imaging studies were performed in a rat model of carotid artery thrombosis. For FBP1 and FBP2 at 120 min post injection, the vessel containing the thrombus showed the highest concentration of radioactivity after the excretory organs, that is, the liver and kidneys. This was confirmed ex vivo by autoradiography, which showed >4-fold activity in the thrombus-containing artery compared to the contralateral artery. FBP3 showed much lower thrombus uptake, and the difference was traced to greater metabolism of this probe. Hybrid MR-PET imaging with FBP1 or FBP2 confirmed that these probes were effective for the detection of an arterial thrombus in this rat model. A thrombus was visible on PET images as a region of high activity that corresponded to a region of arterial occlusion identified by simultaneous MR angiography. FBP1 and FBP2 represent promising new probes for the molecular imaging of thrombi.