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Reducing suffering in animal models and procedures involving seizures, convulsions and epilepsy.
This report is based on discussions and submissions from an expert working group consisting of veterinarians, animal care staff and scientists with expert knowledge relevant to the field and aims to facilitate the implementation of the Three Rs (replacement, reduction and refinement) in the use of animal models or procedures involving seizures, convulsions and epilepsy. Each of these conditions will be considered, the specific welfare issues discussed, and practical measures to reduce animal use and suffering suggested. The emphasis is on refinement since this has the greatest potential for immediate implementation, and some general issues for refinement are summarised to help achieve this, with more detail provided on a range of specific refinements.
CXC chemokines generate age-related increases in neutrophil-mediated brain inflammation and blood-brain barrier breakdown.
Children are at greater risk than adults of permanent brain damage and mortality following head injury or infection [1-5]. Rodent models have demonstrated a 'window of susceptibility' in young animals during which the brain parenchyma is at greater risk of acute neutrophil-mediated breakdown of the blood-brain barrier [6-7]. The exact mechanism of this age-related susceptibility to brain inflammation has yet to be defined, but animal models have revealed that the potent pro-inflammatory cytokine interleukin-1beta (IL-1beta) initiates an intense acute neutrophil-mediated inflammatory response in the brains of young rats and mice that is not seen in adults [6]. Here, we demonstrate the rapid induction of CXC chemokines (which contain a Cys-X-Cys motif), in particular the cytokine-induced neutrophil chemoattractant CINC-1, following the intracerebral administration of IL-1beta. The CXC chemokines produced a more intense neutrophil response in young rats than in adults. The IL-1beta-induced blood-brain barrier breakdown in young rats could be attenuated by an anti-CINC-1 neutralising antibody. These results show that the immature central nervous system (CNS) is dramatically more susceptible to the chemotactic effects of CXC chemokines. Blocking the CXC chemokine activity associated with brain inflammation inhibits neutrophil-mediated blood-brain barrier damage and represents a significant therapeutic possibility.
Axon damage and repair in multiple sclerosis.
It is well known that within long-standing multiple sclerosis (MS) lesions there is axonal loss but whether it is an early or late event has been more difficult to establish. The use of immunocytochemical methods that reveal axonal end-bulbs is a valuable approach to investigating acute axonal injury in human pathological material. The application of these techniques to multiple sclerosis tissue reveals evidence of axonal injury in acute lesions; the distribution of the end-bulbs in acute and active-chronic lesions is associated with regions of maximal density of infiltrating macrophages. Axon injury within the MS lesion will result in both Wallerian degeneration of the axon and also retrograde degeneration of the cell body. The functional consequences of the axon injury will depend upon numbers of axons injured and the topographical organization of the fibres coursing through the lesion. The molecular mechanisms by which the recruited leucocytes damage or transect the axons are not known. However, investigations in the Wld mutant mouse with very slow Wallerian degeneration demonstrate that axon degeneration is not simply a passive disintegration of the axon but has clear parallels with the active processes of programmed cell death. The presence of early axon injury and the consequences of an ever increasing load of neuronal damage has important implications not only for when therapy should be initiated in MS but also the therapeutic target.
The CRTC1-SIK1 pathway regulates entrainment of the circadian clock.
Retinal photoreceptors entrain the circadian system to the solar day. This photic resetting involves cAMP response element binding protein (CREB)-mediated upregulation of Per genes within individual cells of the suprachiasmatic nuclei (SCN). Our detailed understanding of this pathway is poor, and it remains unclear why entrainment to a new time zone takes several days. By analyzing the light-regulated transcriptome of the SCN, we have identified a key role for salt inducible kinase 1 (SIK1) and CREB-regulated transcription coactivator 1 (CRTC1) in clock re-setting. An entrainment stimulus causes CRTC1 to coactivate CREB, inducing the expression of Per1 and Sik1. SIK1 then inhibits further shifts of the clock by phosphorylation and deactivation of CRTC1. Knockdown of Sik1 within the SCN results in increased behavioral phase shifts and rapid re-entrainment following experimental jet lag. Thus SIK1 provides negative feedback, acting to suppress the effects of light on the clock. This pathway provides a potential target for the regulation of circadian rhythms.
Expanding the diversity of chemical protein modification allows post-translational mimicry.
One of the most important current scientific paradoxes is the economy with which nature uses genes. In all higher animals studied, we have found many fewer genes than we would have previously expected. The functional outputs of the eventual products of genes seem to be far more complex than the more restricted blueprint. In higher organisms, the functions of many proteins are modulated by post-translational modifications (PTMs). These alterations of amino-acid side chains lead to higher structural and functional protein diversity and are, therefore, a leading contender for an explanation for this seeming incongruity. Natural protein production methods typically produce PTM mixtures within which function is difficult to dissect or control. Until now it has not been possible to access pure mimics of complex PTMs. Here we report a chemical tagging approach that enables the attachment of multiple modifications to bacterially expressed (bare) protein scaffolds: this approach allows reconstitution of functionally effective mimics of higher organism PTMs. By attaching appropriate modifications at suitable distances in the widely-used LacZ reporter enzyme scaffold, we created protein probes that included sensitive systems for detection of mammalian brain inflammation and disease. Through target synthesis of the desired modification, chemistry provides a structural precision and an ability to retool with a chosen PTM in a manner not available to other approaches. In this way, combining chemical control of PTM with readily available protein scaffolds provides a systematic platform for creating probes of protein-PTM interactions. We therefore anticipate that this ability to build model systems will allow some of this gene product complexity to be dissected, with the aim of eventually being able to completely duplicate the patterns of a particular protein's PTMs from an in vivo assay into an in vitro system.
Molecular MRI enables early and sensitive detection of brain metastases.
Metastasis to the brain is a leading cause of cancer mortality. The current diagnostic method of gadolinium-enhanced MRI is sensitive only to larger tumors, when therapeutic options are limited. Earlier detection of brain metastases is critical for improved treatment. We have developed a targeted MRI contrast agent based on microparticles of iron oxide that enables imaging of endothelial vascular cell adhesion molecule-1 (VCAM-1). Our objectives here were to determine whether VCAM-1 is up-regulated on vessels associated with brain metastases, and if so, whether VCAM-1-targeted MRI enables early detection of these tumors. Early up-regulation of cerebrovascular VCAM-1 expression was evident on tumor-associated vessels in two separate murine models of brain metastasis. Metastases were detectable in vivo using VCAM-1-targeted MRI 5 d after induction (<1,000 cells). At clinical imaging resolutions, this finding is likely to translate to detection at tumor volumes two to three orders of magnitude smaller (0.3-3 × 10(5) cells) than those volumes detectable clinically (10(7)-10(8) cells). VCAM-1 expression detected by MRI increased significantly (P < 0.0001) with tumor progression, and tumors showed no gadolinium enhancement. Importantly, expression of VCAM-1 was shown in human brain tissue containing both established metastases and micrometastases. Translation of this approach to the clinic could increase therapeutic options and change clinical management in a substantial number of cancer patients.
The role of PPAR activation during the systemic response to brain injury.
BACKGROUND: Fenofibrate, a PPAR-α activator, has shown promising results as a neuroprotective therapy, with proposed anti-inflammatory and anti-oxidant effects. However, it displays poor blood-brain barrier permeability leading to some ambiguity over its mechanism of action. Experimentally induced brain injury has been shown to elicit a hepatic acute phase response that modulates leukocyte recruitment to the injured brain. Here, we sought to discover whether one effect of fenofibrate might include the suppression of the acute phase response (APR) following brain injury. METHODS: A 1-h intraluminal thread middle cerebral artery occlusion (MCAO) model followed by a 6-h reperfusion was performed in C57/BL6 mice. Quantitative reverse transcriptase-polymerase chain reaction was then used to measure hepatic expression of chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine ligand 10 (CXCL10) and serum amyloid A-1 (SAA-1), and immunohistochemical analysis was used to quantify brain and hepatic neutrophil infiltration following stroke. RESULTS: The MCAO and sham surgery induced the expression of all three acute phase reactants. A 14-day fenofibrate pre-treatment decreased reactant production, infarct volume, and neutrophil recruitment to the brain and liver, which is a hallmark of the APR. CONCLUSIONS: The data highlight a novel mechanism of action for fenofibrate and lend further evidence towards the promotion of its use as a prophylactic therapy in patients at risk of cerebral ischaemia. Further research is required to elucidate the mechanistic explanation underlying its actions.
A type 2 biomarker separates relapsing-remitting from secondary progressive multiple sclerosis.
OBJECTIVE: We tested whether it is possible to differentiate relapsing-remitting (RR) from secondary progressive (SP) disease stages in patients with multiple sclerosis (MS) using a combination of nuclear magnetic resonance (NMR) metabolomics and partial least squares discriminant analysis (PLS-DA) of biofluids, which makes no assumptions on the underlying mechanisms of disease. METHODS: Serum samples were obtained from patients with primary progressive MS (PPMS), SPMS, and RRMS; patients with other neurodegenerative conditions; and age-matched controls. Samples were analyzed by NMR and PLS-DA models were derived to separate disease groups. RESULTS: The PLS-DA models for serum samples from patients with MS enabled reliable differentiation between RRMS and SPMS. This approach also identified significant differences between the metabolite profiles of each of the MS groups (PP, SP, and RR) and the healthy controls, as well as predicting disease group membership with high specificity and sensitivity. CONCLUSIONS: NMR metabolomics analysis of serum is a sensitive and robust method for differentiating between different stages of MS, yielding diagnostic markers without a priori knowledge of disease pathogenesis. Critically, this study identified and validated a type II biomarker for the RR to SP transition in patients with MS. This approach may be of considerable benefit in categorizing patients for treatment and as an outcome measure in future clinical trials. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that serum metabolite profiles accurately distinguish patients with different subtypes and stages of MS.
Differential induction of interleukin-1beta and tumour necrosis factor-alpha may account for specific patterns of leukocyte recruitment in the brain.
In peripheral tissue, IL-1beta has been shown to induce TNFalpha expression and vice versa, resulting in mixed neutrophil and mononuclear cell recruitment to the site of injury. This has led to the concept of crosstalk in peripheral cytokine signalling pathways. In the brain parenchyma, however, restricted patterns of leukocyte recruitment following the focal injection of pro-inflammatory agents into the brain are observed. This study investigates the expression of the principal pro-inflammatory cytokines--IL-1beta and TNFalpha--in the brain after IL-1beta, TNFalpha, NMDA or endotoxin injection into the brain parenchyma of rats. Each of these agents gives rise to a distinct pattern of acute leukocyte recruitment at 24 h. We found that IL-1beta induces de novo synthesis of additional IL-1beta but not TNFalpha, as determined by RT-PCR and ELISA, and TNFalpha does not induce either itself or IL-1beta. Injection of NMDA results in IL-1beta, but not TNFalpha up-regulation. Injection of IL-1beta or NMDA is associated with neutrophil recruitment whereas injection of TNFalpha is associated with mononuclear cell recruitment. Following injection of endotoxin, both TNFalpha and IL-1beta levels are elevated and neutrophils and mononuclear cells are recruited to the brain. These data suggest that the signalling pathways that are present in the periphery are modified in the brain and that differential induction of TNFalpha and IL-1beta may have a role in the atypical pattern of leukocyte recruitment observed in the brain.
The role of hemorrhage following spinal-cord injury.
Spinal-cord injury is characterized by primary damage as a direct consequence of mechanical insult, and secondary damage that is partly due to the acute inflammatory response. The extent of any hemorrhage within the injured cord is also known to be associated with the formation of intraparenchymal cavities and has been anecdotally linked to secondary damage. This study was designed to examine the contribution of blood components to the outcome of spinal-cord injury. We stereotaxically microinjected collagenase, which causes localized bleeding, into the spinal cord to model the hemorrhage associated with spinal cord injury in the absence of significant mechanical trauma. Tissue damage was observed at the collagenase injection site over time, and was associated with localized disruption of the blood-spinal-cord barrier, neuronal cell death, and the recruitment of leukocytes. The magnitude of the bleed was related to neutrophil mobilization. Interestingly, the collagenase-induced injury also provoked extended axonal damage. With this model, the down-stream effects of hemorrhage are easily discernible, and the impact of treatment strategies for spinal-cord injury on hemorrhage-related injury can be evaluated.
Study of cytokine induced neuropathology by high resolution proton NMR spectroscopy of rat urine.
Multiple sclerosis is a major cause of non-traumatic neurological disability. The identification of markers that differentiate disease progression is critical to effective therapy. A combination of NMR spectroscopic metabolic profiling of urine and statistical pattern recognition was used to detect focal inflammatory central nervous system (CNS) lesions induced by microinjection of a replication-deficient recombinant adenovirus expressing TNF-alpha or IL1-beta cDNA into the brains of Wistar rats. These animals were compared with a group of naïve rats and a group of animals injected with an equivalent null adenovirus. Urine samples were collected 7 days after adenovirus injection, when the inflammatory lesion is maximally active. Principal components analysis and Partial Least Squares-Discriminate analysis of the urine (1)H NMR spectra revealed significant differences between each of the cytokine adenovirus groups and the control groups; for the TNF-alpha group the main differences lay in citrate and succinate, while for the IL-1beta group the predominant changes occurred in leucine, isoleucine, valine and myo-inositol. Thus, we can identify urinary metabolic vectors that not only separate rats with inflammatory lesions in the brain from control animals, but also distinguish between different types of CNS inflammatory lesions.
The murine Cyp1a1 gene is expressed in a restricted spatial and temporal pattern during embryonic development.
In adult mice the cytochrome P450 Cyp1a1 gene is not constitutively expressed but is highly inducible by foreign compounds acting through the aryl hydrocarbon (Ah) receptor. However, the expression profile of the Cyp1a1 gene in the developing embryo is not well under-stood. Using established transgenic mouse lines where 8.5 kb of the rat CYP1A1 promoter is cloned upstream of the lacZ reporter gene (1), we describe the expression of the CYP1A1-driven reporter gene in all tissues through-out stages E7-E14 of embryonic development. In contrast to the absence of constitutive Cyp1a1 and lacZ transgene expression in tissues of the adult mouse, a constitutive cell-specific and time-dependent pattern of CYP1A1 promoter activity was observed in the embryo. This expression pattern was confirmed as reflecting the endogenous gene by measuring Cyp1a1 mRNA levels and protein expression by immunohistochemistry. The number of cells displaying endogenous CYP1A1 activity could be increased in the embryo upon xenobiotic challenge, but only within areas where the CYP1A1 promotor was already active. When reporter mice were bred onto a genetic background expressing a lower affinity form of the Ah receptor (DBA allele), transgene and murine Cyp1a1 protein expression were both attenuated in the adult mouse liver upon xenobiotic challenge. By comparison, constitutive CYP1A1 promoter activity in the embryo was identical in the presence of either the high or low affinity Ah receptor. These novel data suggest that the Cyp1a1 protein may play a role in murine development and that regulation of the Cyp1a1 gene during this period is either through the action of a high affinity Ah receptor ligand or by an alternative regulatory pathway.
[The evidence for primary axonal loss in multiple sclerosis].
INTRODUCTION: At what stage in the pathogenesis of multiple sclerosis (MS) does the damage to axons occur, and why should there be any axon loss at all in what is thought to be principally an axon sparing demyelinating disease? A recently described new technique for investigating axon damage depends for its ability on the immunoreactivity of amiloid precursor protein (APP), which has been shown to be more sensitive than silver stains for detecting damaged axons. DEVELOPMENT: We used APP immunoreactivity as a method to investigate whether axon damage occurs in acute MS lesions. The results of our APP staining showed that the expression of APP in MS lesions is associated with acute MS lesions and the active border of less acute lesions. There was little, if any, APP expression in the chronic lesions. If we accept that the APP staining represents irreversible damage to some axons, the next question is what factors are responsible for mediating damage to axons in MS? Matrix metalloproteinases (MMP) are expressed by macrophages in acute MS lesions and in the active borders of active chronic lesions. The injection of highly-purified MMP into the brain results in demyelination, blood-brain barrier breakdown, and axonal loss. Moreover, the inhibition of the MMP activity reduces the severity of MS-like lesions in experimental models. Thus the properties and distribution of these enzymes make them rational targets for therapeutic intervention. CONCLUSION: Whatever mechanism proves to be responsible for axonal damage in MS, it is clear that this disease should, perhaps, be more appropriately recognized as a primary demyelinating entity with associated primary axonal loss.
The evidence for primary axonal loss in multiple sclerosis
Introduction. At what stage in the pathogenesis of multiple sclerosis (MS) does the damage to axons occur, and why should there be any axon loss at all in what is thought to be principally an axon sparing demyelinating disease? A recently described new technique for investigating axon damage depends for its ability on the immunoreactivity of amiloid precursor protein (APP), which has been shown to be more sensitive than silver stains for detecting damaged axons. Development. We used APP immunore-activity as a method to investigate whether axon damage occurs in acute MS lesions. The results o four APP staining showed that the expression of APP in MS lesions is associated with acute MS lesions and the active border of less acute lesions. There was little, if any, APP expression in the chronic lesions. If we accept that the APP staining represents irreversible damage to some axons, the next question is what factors are responsible for mediating damage to axons in MS? Matrix metalloproteinases (MMP) are expressed by macrophages in acute MS lesions and in the active borders of active chronic lesions. The injection of highly-purified MMP into the brain results in demyelination, blood-brain barrier breakdown, and axonal loss. Moreover, the inhibition of the MMP activity reduces the severity of MS-like lesions in experimental models. Thus the properties and distribution of these enzymes make them rational targets for therapeutic intervention. Conclusion. Whatever mechanism proves to be responsible for axonal damage in MS, it is clear that this disease should, perhaps, be more appropriately recognized as a primary demyelinating entity with associated primary axonal loss.
The effect of B-cell depletion in the Theiler's model of multiple sclerosis.
B cell depletion (BCD) is being considered as a treatment for multiple sclerosis (MS), but there are many uncertainties surrounding the use of this therapy, such as its potential effect in individuals with concurrent viral infections. We sought to discover what effect BCD, induced by an anti-CD20 monoclonal antibody, would have on Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Mice were injected with the anti-CD20 monoclonal antibody 5D2, 14 days before or 14 days after infection with TMEV. Efficacy of depletion of B cells was assessed by flow cytometry of CD19(+) cells. Mouse disability was measured by Rotarod, viral load was measured by real time PCR for TMEV RNA. Binding and neutralizing antibody levels were determined in sera and CSF by ELISA, and in CNS by real time PCR for IgG RNA. Inflammation, microglial activation, axonal damage and demyelination were assessed using immunohistochemistry. 5D2-induced BCD was confirmed by demonstration of nearly absent CD19(+) cells in the blood and lymphoid tissue. Systemic and CNS antibody responses were suppressed during 5D2 treatment. Higher viral loads were detected in 5D2-treated mice than in controls, and the viral levels correlated negatively with IgG production in the brain. Overall, 5D2 caused worsening of the early encephalitis and faster progression of disability, as well as exacerbation of the pathology of TMEV-IDD at the end stage of the disease. These data indicate that BCD in humans might worsen CNS viral infections and might not improve disability accrual in MS.