Felix P. Eckenstein

Prevention of age-related spatial memory deficits in a transgenic mouse model of Alzheimer's disease by chronic Ginkgo biloba treatment.

Alzheimers disease (AD) is characterized by cognitive decline and deposition of beta-amyloid (Abeta) plaques in cortex and hippocampus. A transgenic mouse AD model (Tg2576) that overexpresses a mutant form of human Abeta precursor protein exhibits age-related cognitive deficits, Abeta plaque deposition, and oxidative damage in the brain. We tested the ability of Ginkgo biloba, a flavonoid-rich antioxidant, to antagonize the age-related behavioral impairment and neuropathology exhibited by Tg2576 mice. At 8 months of age, 16 female Tg2576 and 15 female wild-type (wt) littermate mice were given ad lib access to tap water or Ginkgo biloba (70 mg/kg/day in water). After 6 months of treatment, all mice received Morris water maze training (4 trials/day for 10 days) to assess hippocampal dependent spatial learning. All mice received a 60-s probe test of spatial memory retention 24 h after the 40th trial. Untreated Tg2576 mice exhibited a spatial learning impairment, relative to wt mice, while Ginkgo biloba-treated Tg2576 mice exhibited spatial memory retention comparable to wt during the probe test. Spatial learning was not different between Ginkgo biloba-treated and untreated wt mice. There were no group differences in learning to swim to a visible platform. Soluble Abeta and hippocampal Abeta plaque burden did not differ between the Tg2576 groups. Brain levels of protein carbonyls were paradoxically elevated in Ginkgo biloba-treated mice. These data indicate that chronic Ginkgo biloba treatment can block an age-dependent decline in spatial cognition without altering Abeta levels and without suppressing protein oxidation in a transgenic mouse model of AD.

Monofilament intraluminal middle cerebral artery occlusion in the mouse

The rat middle cerebral artery (MCA) occlusion model with an intraluminal filament is well characterized with a two hour period of occlusion in widespread use. The recent availability of transgenic animals has led to an interest in adapting the MCA model in the mouse. To date the model has not been well characterized in the mouse. We performed the present study to compare different durations of MCA occlusion and to validate new functional assessments in this model. The MCA occlusion model (5-0 filament) was used. Swiss-Webster mice, 24-44 g, were randomly assigned to four groups: one hour of occlusion; two hours of occlusion; three hours of occlusion; or permanent occlusion. At 48 hours post-ischemia, the animals were rated on three neurologic function scales, and then the brains were removed for lesion size determination. Overall, there was a significant difference in lesion volume (p < 0.001) between the groups. In the permanent group of mice, the average lesion volume was 78.41 +/- 17.47 mm (n = 12); two and three hours of ischemia produced 51.29 +/- 29.82 mm3 (n = 11) and 54.85 mm3 (n = 13), respectively, significantly different than the one hour group 14.84 +/- 31.34 mm3 (n = 11). All three functional scoring systems found significant overall differences between the four groups with our detailed General and Focal scores producing more robust between group treatment differences and showing correlation coefficients of r = 0.766 and r = 0.788, respectively to infarct volume. The MCA filament occlusion model can be successfully adapted in the mouse with either two or three hour occlusions producing reliable infarcts. New functional scoring systems unique to the mouse appear to add additional information.

Lack of Interleukin-6 Expression Is Not Protective Against Focal Central Nervous System Ischemia

BACKGROUND AND PURPOSE Interleukin-6 (IL-6) appears to be involved in the inflammatory response associated with central nervous system (CNS) ischemia. Although IL-6 levels increase after stroke, it is not known whether IL-6 directly influences CNS ischemic injury. In this study, we used a focal reversible stroke model to investigate whether mice lacking IL-6 were protected against acute ischemic injury. METHODS We bred IL-6-deficient C57 black mice (I-129 IL-6 KO back-crossed with C57), including homozygous knockouts (IL-6 -/-), heterozygous littermates (IL-6 +/-), and normal littermates (IL-6 +/+). The status of all animals was confirmed by DNA sampling and polymerase chain reaction analysis. Reversible middle cerebral artery occlusion was produced by advancing a silicone-coated 8-0 filament into the internal carotid artery for 2 hours (experiment 1) or 45 minutes (experiment 2). At 24 hours, animals were evaluated on a 28-point clinical scale, blood and cerebrospinal fluid were obtained, and the brains were evaluated for infarct volume and IL-6 mRNA levels. RESULTS In experiment 1 (severe ischemia), no differences were seen in lesion size or neurological function between the groups: lesion volume was IL-6 -/- (n=15), 57+/-13 mm(3); IL-6 +/- (n=15), 58+/-23 mm(3); and IL-6 +/+ (n=15), 58+/-18 mm(3) (P=NS). ELISA testing confirmed very low to absent levels of IL-6 in the serum and cerebrospinal fluid of knockout animals. Brain mRNA levels of the other proinflammatory cytokines, including tumor necrosis factor-alpha, IL-1beta, and IL-1 receptor antagonist, were 50% lower in IL-6-deficient ischemic animals than in normal animals. In experiment 2 (mild ischemia), no differences were seen in lesion size or neurological function between the groups: lesion volume was IL-6 -/- (n=10), 16+/-8 mm(3); IL-6 +/- (n=10), 14+/-4 mm(3); and IL-6 +/+ (n=10), 19+/-12 mm(3) (P=NS). CONCLUSIONS In this study, infarct size and neurological function at 24 hours were not different in animals deficient in IL-6 after transient CNS ischemia. This suggests that IL-6 does not have a direct influence on acute ischemic injury. Further study investigating the role of IL-6 on long-term recovery after stroke is in progress.

Differential patterns of ERK and STAT3 phosphorylation after sciatic nerve transection in the rat.

Peripheral nerve injury induces a specific pattern of expression of growth factors and cytokines, which regulate injury responses and regeneration. Distinct classes of growth factors and cytokines signal through specific intracellular phosphorylation cascades. For example, the ERK phosphorylation cascade mediates signaling through transmembrane tyrosine kinase receptors and the JAK/STAT cascade mediates signaling through the GP130 receptor complex. We tested whether specific phosphorylation patterns of ERK and STAT3 result from nerve injury and whether such phosphorylation correlates with the expression of specific growth factors and cytokines. At sites adjacent to a nerve transection, we observed that ERK phosphorylation peaked early, persisted throughout 16 days, and was equally intense at proximal and distal sites. In contrast, STAT3 phosphorylation peaked later than ERK but did not persist as long and was stronger in the proximal than in the distal segment adjacent to the injury. In addition, in distal segments further away from the injury site, ERK became phosphorylated with a delayed time course, while STAT3 remained unphosphorylated. These patterns of phosphorylation correlated well with the expression of neurotrophin and interleukin-6 mRNAs in the distal stump. In addition, we found that the pattern of SAPK phosphorylation is similar to the pattern observed for STAT3, while the pattern of macrophage infiltration into the transected nerve was distinct from all the phosphorylation patterns observed. Together, these observations suggest that ERK activation is important in the establishment of a regeneration-promoting extracellular environment in the far distal stump of transected nerves and that STAT3 activation is important in the control of cellular responses close to the site of injury.

Transient expression of NGF-receptor-like immunoreactivity in postnatal rat brain and spinal cord ☆

The pattern of expression of nerve growth factor (NGF)-receptor-like immunoreactivity during postnatal development in rat central nervous system (CNS) was analyzed using immunohistochemical localization of the receptor. Interestingly, in addition to the expected staining in basal forebrain, several structures were strongly labelled only during specific postnatal developmental stages. These structures included fibers in the thalamus, the external granule cell layer of the cerebellum and motor neurons, indicating that specific neurotrophic mechanisms might play an important role for the labelled cells during a precisely defined period.

Purification and characterization of a trophic factor for embryonic peripheral neurons: Comparison with fibroblast growth factors

The validation of NGF as a physiologically important neurotrophic factor has led to intense efforts to identify novel polypeptide growth factors for neurons. We report here the details of a greater than 80,000-fold purification of a neurotrophic molecule, referred to as growth-promoting activity (GPA), from chicken sciatic nerves. The final product of the purification migrated as a protein band of 21.5 kd, its apparent pI was approximately 4.8, and the ED50 of the most active preparation was approximately 10 pg/ml. Amino acid sequence of a proteolytic digestion fragment of GPA revealed homology with the recently published sequences for rabbit and rat sciatic nerve CNTF. Thus this molecule may be the chicken form of CNTF. Analysis of the specificity of action of GPA showed that, in addition to E8 ciliary ganglion neurons, the factor was able to support short-term survival of E8 dorsal root ganglion and E12 sympathetic neurons. This range of specificities of biological action was also seen with both acidic and basic FGF in the presence of heparin. The biological activity of GPA differed from that of FGF in that it was not potentiated by heparin and did not stimulate mitogenesis in chick fibroblasts.

Temporal modulation of cytokine expression following focal cerebral ischemia in mice.

There is increasing evidence that the inflammatory response plays an important role in CNS ischemia. The murine model of focal ischemia, however, remains incompletely characterized. In this study we examined expression of several cytokines and the vascular adhesion molecule E-selectin, in order to characterize the molecular events following stroke in the C57BL/6J mouse. Using a multi-probe RNAse protection assay (RPA), mRNA for 19 cytokines was analyzed following permanent and transient occlusion of the middle cerebral artery in mice. In addition, samples from the same mice were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate E-selectin mRNA expression levels. Several cytokine mRNAs showed a similar expression pattern in both permanent and transient CNS ischemia while others showed a temporal expression pattern that was dependent on the type of stroke. For both models, mRNA levels of TNFalpha rose early (4 h) followed by IL-6 (10-18 h) and a comparatively late increase (96 h) in TGFbeta1. IL-1alpha, IL-1beta and IL-1ra levels showed a model dependent shift in temporal expression. Reperfusion appeared to delay the induction of these cytokines. Temporal changes in cytokine mRNA expression in the mouse CNS occur following ischemic damage. Our findings demonstrate the utility and power of multi-probe RPA for evaluation of changes in cytokine mRNA levels. Moreover, this study is, to our knowledge the first to show temporal changes in cytokine mRNA in mouse cerebral ischemia, forming a basis for further exploration of the roles of these cytokines in modulating ischemic neuronal damage in this model.

Cloning, expression during development, and evidence for release of a trophic factor for ciliary ganglion neurons

Ciliary ganglion (CG) neurons undergo a period of cell death during development that may be regulated by the limited availability of trophic factor produced by their target tissues. We have previously reported the purification of a ciliary neurotrophic factor from adult chick sciatic nerve that we called growth promoting activity (GPA). Here we demonstrate that GPA can be purified and cloned from embryonic day 15 (E15) chick eyes, which contain all the target tissues of the CG. Our studies show the following: GPA mRNA is induced in embryonic chick eyes during the period of CG neuron cell death; GPA mRNA is expressed specifically in the layer of the eye that contains the targets of the CG and in primary cultures of smooth muscle cells isolated from the choroid layer of the eye; and biologically active GPA is released from cells transfected with a GPA cDNA.

Induction of somatostatin immunoreactivity in cultured ciliary ganglion neurons by activin in choroid cell-conditioned medium

We have previously shown that the expression of somatostatin-like immunoreactivity in cultured ciliary ganglion neurons is stimulated by a macromolecule found in choroid cell-conditioned medium (ChCM). Here, we present the following evidence that this somatostatin-stimulating activity (SSA) is activin: human recombinant activin induces somatostatin-like immunoreactivity in CG neurons; ChCM induces hemoglobin synthesis in K562 cells, a biological activity characteristic of activin; activin A-specific antibodies recognize a protein in ChCM; cultured choroid cells contain activin RNA; and SSA is inhibited by follistatin, a specific activin-binding protein. Thus, activin is likely to be a neurodifferentiation factor for CG neurons in vivo.

Time course of IL-6 expression in experimental CNS ischemia.

Interleukin-6 (IL-6) appears to be an important modulator of the inflammatory response associated with CNS ischemia. Clinically, IL-6 values obtained in the first week post-stroke have been shown to correlate with infarct size and outcome. In this study we used a focal reversible stroke model to investigate the time course and relationship to outcome of IL-6 production in plasma, brain and CSF. Reversible middle cerebral artery occlusion or sham surgery was produced in 50 adult Swiss Webster mice by advancing an 8-0 filament into the internal carotid artery for 2 h (sham 1 min). At 3, 6, 12, 24, and 72 h (8 each ischemia; 2 each sham) groups of animals were evaluated on a 28 point clinical scale, blood and CSF obtained, and the brains were evaluated for infarct volume and IL-6 mRNA levels. Serum levels of IL-6 (ELISA mean +/- SD; undetectable in controls) overall sham group, 102 +/- 87; 3 h, 908 +/- 494* pg ml-1; 6 h, 1079 +/- 468* pg ml-1; 12 h, 980 +/- 221* pg ml-1; pg ml-1; 24 h, 320 +/- 314* pg ml-1; 72 h, 20 +/- 30* pg ml-1 (*p < or = 0.05 to sham). CSF levels (ELISA) overall sham group, 10 +/- 18; 3 h, 379 +/- 210* pg ml-1; 6 h, 157 +/- 61* pg ml-1; 12 h, 136 +/- 88* pg ml-1; 24 h, 127 +/- 99 pg ml-1; 72 h, 72 +/- 9* pg ml-1 (*p < or = 0.05 to sham). Brain IL-6 mRNA levels overall sham group, 20; 3 h, 480; 6 h, 599; 12 h, 7960; 24 h, 20267; 72 h, 0. There was an overall R2 of 0.20 between plasma and CSF IL-6. There was an overall R2 of 0.13 and 0.20 between infarct size and serum and CSF IL-6 level respectively, and an overall R2 of 0.10 and 0.17 between neurologic function and serum and CSF IL-6 level respectively. These findings confirm that IL-6 values increase following CNS ischemia with peak serum and CSF levels occurring before brain values. CSF IL-6 levels had a stronger correlation with neurologic function and infarct size than serum.