Molecular alterations of the TLR4-signaling cascade in canine epilepsy.

BACKGROUND: Cumulating evidence from rodent models points to a pathophysiological role of inflammatory signaling in the epileptic brain with Toll-like receptor-4 signaling acting as one key factor. However, there is an apparent lack of information about expression alterations affecting this pathway in canine patients with epilepsy. Therefore, we have analyzed the expression pattern of Toll-like receptor 4 and its ligands in brain tissue of canine patients with structural or idiopathic epilepsy in comparison with tissue from laboratory dogs or from owner-kept dogs without neurological diseases. RESULTS: The analysis revealed an overexpression of Toll-like receptor-4 in the CA3 region of dogs with structural epilepsy. Further analysis provided evidence for an upregulation of Toll-like receptor-4 ligands with high mobility group box-1 exhibiting increased expression levels in the CA1 region of dogs with idiopathic and structural epilepsy, and heat shock protein 70 exhibiting increased expression levels in the piriform lobe of dogs with idiopathic epilepsy. In further brain regions, receptor and ligand expression rates proved to be either in the control range or reduced below control levels. CONCLUSIONS: Our study reveals complex molecular alterations affecting the Toll-like receptor signaling cascade, which differ between epilepsy types and between brain regions. Taken together, the data indicate that multi-targeting approaches modulating Toll-like receptor-4 signaling might be of interest for management of canine epilepsy. Further studies are recommended to explore respective molecular alterations in more detail in dogs with different etiologies and to confirm the role of the pro-inflammatory signaling cascade as a putative target.

Defining body-weight reduction as a humane endpoint: a critical appraisal.

In many animal experiments scientists and local authorities define a body-weight reduction of 20% or more as severe suffering and thereby as a potential parameter for humane endpoint decisions. In this study, we evaluated distinct animal experiments in multiple research facilities, and assessed whether 20% body-weight reduction is a valid humane endpoint criterion in rodents. In most experiments (restraint stress, distinct models for epilepsy, pancreatic resection, liver resection, caloric restrictive feeding and a mouse model for Dravet syndrome) the animals lost less than 20% of their original body weight. In a glioma model, a fast deterioration in body weight of less than 20% was observed as a reliable predictor for clinical deterioration. In contrast, after induction of chronic diabetes or acute colitis some animals lost more than 20% of their body weight without exhibiting major signs of distress. In these two animal models an exclusive application of the 20% weight loss criterion for euthanasia might therefore result in an unnecessary loss of animals. However, we also confirmed that this criterion can be a valid parameter for defining the humane endpoint in other animal models, especially when it is combined with additional criteria for evaluating distress. In conclusion, our findings strongly suggest that experiment and model specific considerations are necessary for the rational integration of the parameter 'weight loss' in severity assessment schemes and humane endpoint criteria. A flexible implementation tailored to the experiment or intervention by scientists and authorities is therefore highly recommended.

Impact of repeated kindled seizures on heart rate rhythms, heart rate variability, and locomotor activity in rats.

Although an impact of epilepsy on circadian rhythmicity is well-recognized, there are profound gaps in our understanding of the influence of seizures on diurnal rhythms. The effect on activity levels and heart rate is of particular interest as it might contribute to the disease burden. The kindling model with telemetric transmitter implants provides excellent opportunities to study the consequences of focal and generalized seizures under standardized conditions. Data from kindled rats with generalized seizures revealed an increase in activity and heart rate during the resting phase. Total and short-term heart rate variabilities were not affected by electrode implantation or seizure induction. Ictal alterations in heart rate associated with generalized seizures were characterized by a biphasic bradycardia with an immediate drop of heart rate followed by a transient normalization and a second more steady decrease. In conclusion, the findings demonstrate that once daily generalized seizures can exert significant effects on heart rate rhythms. Respective alterations in patients would be of relevance for patient counselling and therapeutic management. Occurrence of biphasic bradycardia associated with seizure induction suggests that the kindling model is suitable to study the consequences and the prevention of ictal bradycardia, which may pose patients at risk for sudden unexpected death.

Genetic and Pharmacological Targeting of Heat Shock Protein 70 in the Mouse Amygdala-Kindling Model.

Inflammatory responses involving Toll-like receptor signaling represent a key factor contributing to epileptogenesis. Thus, it is of particular interest to explore the relevance of toll-like receptor ligands and modulators, such as heat shock protein 70 (HSP70). Motivated by recent findings demonstrating an upregulation of HSP70 in a model of epileptogenesis, we analyzed the consequences of genetic and pharmacological targeting of HSP70 expression in a mouse kindling paradigm. Lack of inducible HSP70 resulted in increased prekindling seizure thresholds. However, at threshold stimulation the deficiency-promoted seizure spread, as indicated by an increased seizure severity. Subsequent kindling stimulations elicited more severe seizures in knockout mice, whereas endogenous termination of seizure activity remained unaffected with duration of behavioral and electrographic seizure activity comparable to that of wild-type mice. Interestingly, HSP70 deficiency resulted in enhanced microglia activation in the CA1 region. Next, we assessed a pharmacological targeting approach aiming to promote HSP70 expression. Celastrol treatment had no impact on kindling progression but reduced postkindling seizure thresholds and enhanced microglia activation in CA1 and CA3. In conclusion, the findings from HSP70-knockout mice support a protective role of HSP70 with an effect on microglia activation and spread of seizure activity. Unexpectedly, celastrol administration resulted in detrimental consequences. These findings should be considered as a warning about the general safety of celastrol as a drug candidate. The impact of pathophysiological mechanisms on the quality of celastrol effects requires comprehensive future studies exploring influencing factors. Moreover, alternate strategies to increase HSP70 expression should be further developed and validated.

Genetic Modulation of HSPA1A Accelerates Kindling Progression and Exerts Pro-convulsant Effects.

Strong evidence exists that Toll-like receptor (TLR)-mediated effects on microglia functional states can promote ictogenesis and epileptogenesis. So far, research has focused on the role of high-mobility group box protein 1 as an activator of TLRs. However, the development of targeting strategies might need to consider a role of additional receptor ligands. Considering the fact that heat shock protein A1 (hsp70) has been confirmed as a TLR 2 and 4 ligand, we have explored the consequences of its overexpression in a mouse kindling paradigm. The genetic modulation enhanced seizure susceptibility with lowered seizure thresholds prior to kindling. In contrast to wildtype (WT) mice, HSPA1A transgenic (TG) mice exhibited generalized seizures very early during the kindling paradigm. Along with an increased seizure severity, seizure duration proved to be prolonged in TG mice during this phase. Toward the end of the stimulation phase seizure parameters of WT mice reached comparable levels. However, a difference between genotypes was still evident when comparing seizure parameters during the post-kindling threshold determination. Surprisingly, HSPA1A overexpression did not affect microglia activation in the hippocampus. In conclusion, the findings demonstrate that hsp70 can exert pro-convulsant effects promoting ictogenesis in naïve animals. The pronounced impact on the response to subsequent stimulations gives first evidence that genetic HSPA1A upregulation may also contribute to epileptogenesis. Thus, strategies inhibiting hsp70 or its expression might be of interest for prevention of seizures and epilepsy. However, conclusions about a putative pro-epileptogenic effect of hsp70 require further investigations in models with development of spontaneous recurrent seizures.

Toward evidence-based severity assessment in rat models with repeated seizures: I. Electrical kindling.

OBJECTIVE: Rodent epilepsy models can significantly contribute to our understanding of pathophysiological mechanisms and to validation of biomarker and target candidates. Evidence-based severity assessment is a presupposition for the ethical evaluation of animal experimentation allowances as well as for the development of efficacious refinement concepts. METHODS: Aiming to improve our understanding of the impact of experimental procedures and repeated seizures, we have completed a comprehensive behavioral and biochemical analysis assessing various parameters that can inform about the influence of an electrical kindling paradigm on well-being in rats. Thereby, we have focused on the immediate effects of phases with focal and generalized seizures with behavioral testing during kindling acquisition. RESULTS: Electrode implantation exerted mild effects on anxiety-associated behavior and reduced serum corticosterone at 3 weeks, but not 7 weeks, following surgery. Analysis in kindled rats excluded any relevant impact of focal seizures on behavioral and biochemical parameters. Assessment in rats with generalized seizures revealed an impact on nest complexity scores, nest soiling, and selected parameters in paradigms evaluating anxiety-associated behavior. Moreover, serum corticosterone levels, but neither hair corticosterone nor fecal corticosterone metabolite concentrations were lowered as a consequence of repeated generalized seizures. The assessment of various other behavioral and biochemical parameters did not reveal any other relevant effects of generalized seizures. Cross-correlation analysis suggested that assessment of nest building and maintenance can provide information comparable to that from more elaborate behavioral assays. This finding provides first evidence that nest scoring might serve as a simple and valid approach to evaluate rat well-being during routine assessment schemes. SIGNIFICANCE: The findings argue against a persistent level of pronounced distress and suggest a classification of the kindling paradigm as a model with moderate severity based on a longer-lasting mild impact on animal behavioral patterns. This suggestion provides a basis for a prospective and retrospective case-by-case severity assessment.