P.R. Bär

Exogenous anandamide protects rat brain against acute neuronal injury in vivo

The endocannabinoid anandamide [N-arachidonoylethanolamine (AEA)] is thought to function as an endogenous protective factor of the brain against acute neuronal damage. However, this has never been tested in an in vivo model of acute brain injury. Here, we show in a longitudinal pharmacological magnetic resonance imaging study that exogenously administered AEA dose-dependently reduced neuronal damage in neonatal rats injected intracerebrally with the Na+/K+-ATPase inhibitor ouabain. At 15 min after injury, AEA (10 mg/kg) administered 30 min before ouabain injection reduced the volume of cytotoxic edema by 43 ± 15% in a manner insensitive to the cannabinoid CB1receptor antagonist SR141716A. At 7 d after ouabain treatment, 64 ± 24% less neuronal damage was observed in AEA-treated (10 mg/kg) rats compared with control animals. Coadministration of SR141716A prevented the neuroprotective actions of AEA at this end point. In addition, (1) no increase in AEA and 2-arachidonoylglycerol levels was detected at 2, 8, or 24 hr after ouabain injection; (2) application of SR141716A alone did not increase the lesion volume at days 0 and 7; and (3) the AEA-uptake inhibitor, VDM11, did not affect the lesion volume. These data indicate that there was no endogenous endocannabinoid tone controlling the acute neuronal damage induced by ouabain. Although our data seem to question a possible role of the endogenous cannabinoid system in establishing a brain defense system in our model, AEA may be used as a structural template to develop neuroprotective agents.

Sexual differences in onset of disease and response to exercise in a transgenic model of ALS

Transgenic mice that overexpress the mutant human SOD1 gene (hSOD1) serve as an animal model for amyotrophic lateral sclerosis (ALS). Age and sex are recognized as risk factors for ALS, but physical activity remains controversial. Therefore, we investigated the effect of exercise on the phenotype of male and female hSOD1 mice. Onset of disease, progression of disease and survival were measured in low-copy and high-copy hSOD1 mice that were randomized to an exercise or sedentary group. We found that onset of disease was different for the two sexes: significantly earlier in male than in female hSOD1 mice. Exercise delayed the onset of disease in female but not in male hSOD1 mice. Also, exercise delayed the total survival time in female high-copy hSOD1 mice. Muscle morphometry and motor neuron counts were similar in all experimental groups at the end of training. Sedentary female hSOD1 mice showed more frequently irregular estrous cycles suggesting a higher estrogen exposure in exercising female mice. These results suggest a possible neuroprotective effect of female sex hormones and support the view that ALS patients should not avoid regular exercise.

Collagen Containing Neurotrophin-3 (NT-3) Attracts Regrowing Injured Corticospinal Axons in the Adult Rat Spinal Cord and Promotes Partial Functional Recovery ☆

During development, neurotrophic factors play an important role in the guidance and outgrowth of axons. Our working hypothesis is that neurotrophic factors involved in the development of axons of a particular CNS tract are among the most promising candidates for stimulating and directing the regrowth of fibers of this tract in the lesioned adult animal. The neurotrophin NT-3 is known to be involved in the target selection of outgrowing corticospinal tract (CST) fibers. We studied the capacity of locally applied NT-3 to stimulate and direct the regrowth of axons of the CST in the lesioned adult rat spinal cord. We also studied the effect of NT-3 application on the functional recovery of rats after spinal cord injury, using the gridwalk test. NT-3 was applied at the site of the lesion dissolved into rat tail collagen type I. Four weeks after spinal cord injury and collagen implantation, significantly more CST fibers had regrown into the collagen matrix containing NT-3 (22 +/- 6%, mean +/- SEM) than into the control collagen matrix without NT-3 (7 +/- 2%). No CST fibers grew into areas caudal to the collagen implant. Despite the absence of regrowth of corticospinal axons into host tissue caudal to the lesion area, functional recovery was observed in rats with NT-3 containing collagen implants.

Neuroprotection by the Endogenous Cannabinoid Anandamide and Arvanil against In Vivo Excitotoxicity in the Rat: Role of Vanilloid Receptors and Lipoxygenases

Type 1 vanilloid receptors (VR1) have been identified recently in the brain, in which they serve as yet primarily undetermined purposes. The endocannabinoid anandamide (AEA) and some of its oxidative metabolites are ligands for VR1, and AEA has been shown to afford protection against ouabain-induced in vivo excitotoxicity, in a manner that is only in part dependent on the type 1 cannabinoid (CB1) receptor. In the present study, we assessed whether VR1 is involved in neuroprotection by AEA and by arvanil, a hydrolysis-stable AEA analog that is a ligand for both VR1 and CB1. Furthermore, we assessed the putative involvement of lipoxygenase metabolites of AEA in conveying neuroprotection. Using HPLC and gas chromatography/mass spectroscopy, we demonstrated that rat brain and blood cells converted AEA into 12-hydroxy-N-arachidoylethanolamine (12-HAEA) and 15-hydroxy-N-arachidonoylethanolamine (15-HAEA) and that this conversion was blocked by addition of the lipoxygenase inhibitor nordihydroguaiaretic acid. Using magnetic resonance imaging we show the following: (1) pretreatment with the reduced 12-lipoxygenase metabolite of AEA, 12-HAEA, attenuated cytotoxic edema formation in a CB1 receptor-independent manner in the acute phase after intracranial injection of the Na+/K+-ATPase inhibitor ouabain; (2) the reduced 15-lipoxygenase metabolite, 15-HAEA, enhanced the neuroprotective effect of AEA in the acute phase; (3) modulation of VR1, as tested using arvanil, the VR1 agonist capsaicin, and the antagonist capsazepine, leads to neuroprotective effects in this model, and arvanil is a potent neuroprotectant, acting at both CB1 and VR1; and (4) the in vivo neuroprotective effects of AEA are mediated by CB1 but not by lipoxygenase metabolites or VR1.

Chronic idiopathic polyneuropathy presenting in middle or old age: a clinical and electrophysiological study of 75 patients.

The clinical and electrophysiological features were prospectively studied of 75 patients (46 men and 29 women) with chronic polyneuropathy presenting in middle or old age in whom a diagnosis could not be made even after extensive evaluation and a follow up of six months. The mean age at the onset of symptoms was 56.5 years. The clinical features of chronic idiopathic polyneuropathy are heterogeneous. On clinical grounds 44 patients had a sensorimotor, 29 patients a sensory, and two patients a motor polyneuropathy. The overall clinical course in chronic idiopathic polyneuropathy was slowly progressive. None of the patients became severely disabled. Electrophysiological and nerve biopsy studies were compatible with an axonal polyneuropathy. Antibodies against myelin associated glycoprotein, gangliosides, and sulphatides were assessed in 70 patients and found to be negative.

Acute neuronal injury, excitotoxicity, and the endocannabinoid system.

The endocannabinoid system is a valuable target for drug discovery, because it is involved in the regulation of many cellular and physiological functions. The endocannabinoid system constitutes the endogenous lipids anandamide, 2-arachidonoylglycerol and noladin ether, and the cannabinoid CB1 and CB2 receptors as well as the proteins for their inactivation. It is thought that (endo)cannabinoid-based drugs may potentially be useful to reduce the effects of neurodegeneration. This paper reviews recent developments in the endocannabinoid system and its involvement in neuroprotection.Exogenous (endo)cannabinoids have been shown to exert neuroprotection in a variety of in vitro and in vivo models of neuronal injury via different mechanisms, such as prevention of excitotoxicity by CB1-mediated inhibition of glutamatergic transmission, reduction of calcium influx, and subsequent inhibition of deleterious cascades, TNF-α formation, and anti-oxidant activity. It has been suggested that the release of endogenous endocannabinoids during neuronal injury might be a protective response. However, several observations indicate that the role of the endocannabinoid system as a general endogenous protection system is questionable. The data are critically reviewed and possible explanations are given.

The expression of the chemorepellent Semaphorin 3A is selectively induced in terminal Schwann cells of a subset of neuromuscular synapses that display limited anatomical plasticity and enhanced vulnerability in motor neuron disease

Neuromuscular synapses differ markedly in their plasticity. Motor nerve terminals innervating slow muscle fibers sprout vigorously following synaptic blockage, while those innervating fast-fatigable muscle fibers fail to exhibit any sprouting. Here, we show that the axon repellent Semaphorin 3A is differentially expressed in terminal Schwann cells (TSCs) on different populations of muscle fibers: postnatal, regenerative and paralysis induced remodeling of neuromuscular connections is accompanied by increased expression of Sema3A selectively in TSCs on fast-fatigable muscle fibers. To our knowledge, this is the first demonstration of a molecular difference between TSCs on neuromuscular junctions of different subtypes of muscle fibers. Interestingly, also in a mouse model for amyotrophic lateral sclerosis (ALS), Sema3A is expressed at NMJs of fast-fatigable muscle fibers. We propose that expression of Sema3A by TSCs not only suppresses nerve terminal plasticity at specific neuromuscular synapses, but may also contribute to their early and selective loss in the motor neuron disease ALS.

Interferon-beta prevents cytokine-induced neutrophil infiltration and attenuates blood-brain barrier disruption

Inflammation can contribute to brain injury, such as that resulting from ischemia or trauma. The authors have previously shown that the cytokine interferon-beta (IFN-β) affords protection against ischemic brain injury, which was associated with a diminished infiltration of neutrophils and a reduction in blood–brain barrier (BBB) disruption. The goal of the current study was to directly assess the effects of IFN-β on neutrophil infiltration, with the use of an in vivo assay of neutrophil infiltration with relevance to ischemic brain injury. Intrastriatal injection of recombinant rat cytokine–induced neutrophil chemoattractant-1, a member of the interleukin-8 family (1 μg in 1 μL), triggered massive infiltration of neutrophils and extensive BBB disruption 6 hours later, as measured using immunofluorescence microscopy and magnetic resonance imaging in the rat, respectively. Depleting the animals of neutrophils before interleukin-8 injection prevented BBB disruption. Treatment with IFN-β (5 × 106 U/kg) almost completely prevented neutrophil infiltration and attenuated BBB damage. Gelatinase zymography showed matrix metalloproteinase-9 expression in the ipsilateral striatum after interleukin-8 injection. Both neutrophil depletion and IFN-β treatment downregulated matrix metalloproteinase-9. IFN-β has already been approved for human use as a treatment for the chronic inflammatory disorder multiple sclerosis. The potential value of IFN-β as a treatment that can attenuate acute brain inflammation is considered.

Interferon-Beta Blocks Infiltration of Inflammatory Cells and Reduces Infarct Volume after Ischemic Stroke in the Rat:

The inflammatory response that exacerbates cerebral injury after ischemia is an attractive therapeutic target: it progresses over days and strongly contributes to worsening of the neurologic outcome. The authors show that, after transient ischemic injury to the rat brain, systemic application of interferon-beta (IFN-β), a cytokine with antiinflammatory properties, attenuated the development of brain infarction. Serial magnetic resonance imaging (MRI) showed that IFN-β treatment reduced lesion volume on diffusion-weighted MRI by 70% (P < 0.01) at 1 day after stroke. IFN-β attenuated the leakage of contrast agent through the blood–brain barrier (P < 0.005), indicating a better-preserved blood–brain barrier integrity. Both control and IFN-β-treated animals showed a similar degree of relative hyperperfusion of the lesioned hemisphere, indicating that neuroprotection by IFN-β was not mediated by improved cerebral perfusion as assessed 24 hours after stroke onset. IFN-β treatment resulted in an 85% reduction (P < 0.0001) in infarct volume 3 weeks later, as determined from T2-weighted MRI and confirmed by histology. This effect was achieved even when treatment was started 6 hours after stroke onset. Quantitative immunohistochemistry at 24 hours after stroke onset showed that IFN-β almost completely prevented the infiltration of neutrophils and monocytes into the brain. Gelatinase zymography showed that this effect was associated with a decrease in matrix metalloproteinase-9 expression. In conclusion, treatment with the antiinflammatory cytokine IFN-β affords significant neuroprotection against ischemia/reperfusion injury, and within a relatively long treatment window. Because IFN-β has been approved for clinical use, it may be rapidly tested in a clinical trial for its efficacy against human stroke.

Trophic influences of alpha-MSH and ACTH4–10 on neuronal outgrowth in vitro

Slices of foetal spinal cords in culture were used to establish possible trophic effects of alpha-melanocyte stimulating hormone (alpha-MSH) and a fragment of the adrenocorticotropic hormone (ACTH4-10) on the outgrowth of neurites from spinal neurons. The spinal cord slices were treated with peptides over a wide concentration range. Using monoclonal antibodies against (subunits of) neurofilament followed by immunofluorescence, we could show that the extension consisted mainly of axons. After 5 and 7 days, outgrowth was quantified with 2 different techniques, namely by visual scoring under phase contrast and by means of an ELISA for neurofilament protein. Both methods yielded the same dose-response profile. Both alpha-MSH and ACTH4-10 stimulated the formation of neurites in a dose-dependent manner, with a maximal stimulatory effect at 0.001-0.01 nM (ACTH4-10) or 0.1-1.0 nM (alpha-MSH). The maximal effect of the peptides was 30-40% compared to controls. We conclude that alpha-MSH and ACTH4-10 stimulate axonal outgrowth from foetal spinal cord slices in vitro in a dose-dependent way.