Botulinum and Tetanus Neurotoxins.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most potent toxins known and cause botulism and tetanus, respectively. BoNTs are also widely utilized as therapeutic toxins. They contain three functional domains responsible for receptor-binding, membrane translocation, and proteolytic cleavage of host proteins required for synaptic vesicle exocytosis. These toxins also have distinct features: BoNTs exist within a progenitor toxin complex (PTC), which protects the toxin and facilitates its absorption in the gastrointestinal tract, whereas TeNT is uniquely transported retrogradely within motor neurons. Our increasing knowledge of these toxins has allowed the development of engineered toxins for medical uses. The discovery of new BoNTs and BoNT-like proteins provides additional tools to understand the evolution of the toxins and to engineer toxin-based therapeutics. This review summarizes the progress on our understanding of BoNTs and TeNT, focusing on the PTC, receptor recognition, new BoNT-like toxins, and therapeutic toxin engineering.

Long-term seizure dynamics are determined by the nature of seizures and the mutual interactions between them.

The seemingly random and unpredictable nature of seizures is a major debilitating factor for people with epilepsy. An increasing body of evidence demonstrates that the epileptic brain exhibits long-term fluctuations in seizure susceptibility, and seizure emergence seems to be a consequence of processes operating over multiple temporal scales. A deeper insight into the mechanisms responsible for long-term seizure fluctuations may provide important information for understanding the complex nature of seizure genesis. In this study, we explored the long-term dynamics of seizures in the tetanus toxin model of temporal lobe epilepsy. The results demonstrate the existence of long-term fluctuations in seizure probability, where seizures form clusters in time and are then followed by seizure-free periods. Within each cluster, seizure distribution is non-Poissonian, as demonstrated by the progressively increasing inter-seizure interval (ISI), which marks the approaching cluster termination. The lengthening of ISIs is paralleled by: increasing behavioral seizure severity, the occurrence of convulsive seizures, recruitment of extra-hippocampal structures and the spread of electrographic epileptiform activity outside of the limbic system. The results suggest that repeated non-convulsive seizures obey the 'seizures-beget-seizures' principle, leading to the occurrence of convulsive seizures, which decrease the probability of a subsequent seizure and, thus, increase the following ISI. The cumulative effect of repeated convulsive seizures leads to cluster termination, followed by a long inter-cluster period. We propose that seizures themselves are an endogenous factor that contributes to long-term fluctuations in seizure susceptibility and their mutual interaction determines the future evolution of disease activity.

The role of the single interchains disulfide bond in tetanus and botulinum neurotoxins and the development of antitetanus and antibotulism drugs.

A large number of bacterial toxins consist of active and cell binding protomers linked by an interchain disulfide bridge. The largest family of such disulfide-bridged exotoxins is that of the clostridial neurotoxins that consist of two chains and comprise the tetanus neurotoxins causing tetanus and the botulinum neurotoxins causing botulism. Reduction of the interchain disulfide abolishes toxicity, and we discuss the experiments that revealed the role of this structural element in neuronal intoxication. The redox couple thioredoxin reductase-thioredoxin (TrxR-Trx) was identified as the responsible for reduction of this disulfide occurring on the cytosolic surface of synaptic vesicles. We then discuss the very relevant finding that drugs that inhibit TrxR-Trx also prevent botulism. On this basis, we propose that ebselen and PX-12, two TrxR-Trx specific drugs previously used in clinical trials in humans, satisfy all the requirements for clinical tests aiming at evaluating their capacity to effectively counteract human and animal botulism arising from intestinal toxaemias such as infant botulism.

Cardiac effects of repeated focal seizures in rats induced by intrahippocampal tetanus toxin: Bradyarrhythmias, tachycardias, and prolonged interictal QT interval.

OBJECTIVE: To determine electrical changes in the heart in a chronic, nonstatus model of epilepsy. METHODS: Electrocorticography (ECoG) and electrocardiography (ECG) of nine animals (five made epileptic by intrahippocampal injection of tetanus neurotoxin (TeNT) and four controls), are monitored continuously by radiotelemetry for up to 7 weeks. RESULTS: Epileptic animals develop a median of 168 seizures, with postictal tachycardias reaching a mean of 487 beats/min and lasting a mean of 661 seconds. Ictal changes in heart rate include tachycardia and in the case of convulsive seizures, bradyarrhythmias resembling Mobitz type 1 second-degree atrioventricular block; notably the P-R interval increased before block. Postictally, the amplitude of T wave increases. Interictally, QT dependence on RR is modest and conventional QT corrections prove ineffective. Interictal QT intervals, measured at a heart rate of 400 bpm, increased from 65 to 75 ms, an increase dependent on seizure incidence over the preceding 10-14 days. SIGNIFICANCE: Repeated seizures induce a sustained tachycardia and increase in QT interval of the ECG and evoke arrhythmias including periods of atrioventricular block during Racine type 4 and 5 seizures. These changes in cardiac function may predispose to development in fatal arrhythmias and sudden death in humans with epilepsy.

Partial Raphe Dysfunction in Neurotransmission Is Sufficient to Increase Mortality after Anoxic Exposures in Mice at a Critical Period in Postnatal Development.

Sudden infant death syndrome (SIDS) cases often have abnormalities of the brainstem raphe serotonergic (5-HT) system. We hypothesize that raphe dysfunction contributes to a failure to autoresuscitate from multiple hypoxic events, leading to SIDS. We studied autoresuscitation in two transgenic mouse models in which exocytic neurotransmitter release was impaired via conditional expression of the light chain from tetanus toxin (tox) in raphe neurons expressing serotonergic bacterial artificial chromosome drivers Pet1 or Slc6a4. These used recombinase drivers targeted different portions of medullary raphe serotonergic, tryptophan hydroxylase 2 (Tph2)(+) neurons by postnatal day (P) 5 through P12: approximately one-third in triple transgenic Pet1::Flpe, hßactin::cre, RC::PFtox mice; approximately three-fourths inSlc6a4::cre, RC::Ptox mice; with the first model capturing a near equal number of Pet1(+),Tph2(+) versus Pet1(+),Tph2(low or negative) raphe cells. At P5, P8, and P12, "silenced" mice and controls were exposed to five, ∼37 s bouts of anoxia. Mortality was 5-10 times greater in "silenced" pups compared with controls at P5 and P8 (p = 0.001) but not P12, with cumulative survival not differing between experimental transgenic models. "Silenced" pups that eventually died took longer to initiate gasping (p = 0.0001), recover heart rate (p = 0.0001), and recover eupneic breathing (p = 0.011) during the initial anoxic challenges. Variability indices for baseline breathing distinguished "silenced" from controls but did not predict mortality. We conclude that dysfunction of even a portion of the raphe, as observed in many SIDS cases, can impair ability to autoresuscitate at critical periods in postnatal development and that baseline indices of breathing variability can identify mice at risk. SIGNIFICANCE STATEMENT: Many sudden infant death syndrome (SIDS) cases exhibit a partial (∼26%) brainstem serotonin deficiency. Using recombinase drivers, we targeted different fractions of serotonergic and raphe neurons in mice for tetanus toxin light chain expression, which prevented vesicular neurotransmitter release. In one model, approximately one-third of medullary Tph2(+) neurons are silenced by postnatal (P) days 5 and 12, along with some Pet1(+),Tph2(low or negative) raphe cells; in the other, approximately three-fourths of medullary Tph2(+) neurons, also with some Tph2(low or negative) cells. Both models demonstrated excessive mortality to anoxia (a postulated SIDS stressor) at P5 and P8. We demonstrated fatal vulnerability to anoxic stress at a specific time in postnatal life induced by a partial defect in raphe function. This models features of SIDS.

Single-cell profiling of lineage determining transcription factors in antigen-specific CD4+ T cells reveals unexpected complexity in recall responses during immune reconstitution.

Recent studies of protein and gene expression at the single-cell level have revealed that the memory T-cell compartment is more heterogeneous than previously acknowledged. Identifying different T helper subsets involved in memory responses at the single-cell level is thus necessary to understand the level of heterogeneity within this population. Antigen-specific CD4+ T cells were measured using the CD25/OX40 assay together with a qualitative multiplex single-cell RT-PCR assay. Transcription profiles and subset proportions within the antigen-specific CD4+ T-cell population were dissected. Cytomegalovirus (CMV)-specific CD4+ T-cell responses skewed toward a Th1 response, whereas Tetanus toxoid responses skewed toward a Th2 type response. Fluctuations in CD4+ T-cell subsets were observed within the HIV-Gag-specific response during ongoing antiretroviral therapy. Strong effector responses (Th1) were observed in early treatment, however with ongoing therapy this effector response significantly decreased in combination with an increase in Tregs and circulating Tfh-like BCL-6+ memory cells. The apparent increase in Tcm in peripheral blood after a several weeks of antiretroviral therapy may be due to Tfh-like cell egress from germinal centers into the periphery.

Rapid eye movement sleep and hippocampal theta oscillations precede seizure onset in the tetanus toxin model of temporal lobe epilepsy.

Improved understanding of the interaction between state of vigilance (SOV) and seizure onset has therapeutic potential. Six rats received injections of tetanus toxin (TeTX) in the ventral hippocampus that resulted in chronic spontaneous seizures. The distribution of SOV before 486 seizures was analyzed for a total of 19 d of recording. Rapid eye movement sleep (REM) and exploratory wake, both of which express prominent hippocampal theta rhythm, preceded 47 and 34%, for a total of 81%, of all seizures. Nonrapid eye movement sleep (NREM) and nonexploratory wake, neither of which expresses prominent theta, preceded 6.8 and 13% of seizures. We demonstrate that identification of SOV yields significant differentiation of seizure susceptibilities, with the instantaneous seizure rate during REM nearly 10 times higher than baseline and the rate for NREM less than half of baseline. Survival analysis indicated a shorter duration of preseizure REM bouts, with a maximum transition to seizure at ∼90 s after the onset of REM. This study provides the first analysis of a correlation between SOV and seizure onset in the TeTX model of temporal lobe epilepsy, as well as the first demonstration that hippocampal theta rhythms associated with natural behavioral states can serve a seizure-promoting role. Our findings are in contrast with previous studies suggesting that the correlations between SOV and seizures are primarily governed by circadian oscillations and the notion that hippocampal theta rhythms inhibit seizures. The documentation of significant SOV-dependent seizure susceptibilities indicates the potential utility of SOV and its time course in seizure prediction and control.

Tetanus neurotoxin utilizes two sequential membrane interactions for channel formation.

Tetanus neurotoxin (TeNT) causes neuroparalytic disease by entering the neuronal soma to block the release of neurotransmitters. However, the mechanism by which TeNT translocates its enzymatic domain (light chain) across endosomal membranes remains unclear. We found that TeNT and a truncated protein devoid of the receptor binding domain (TeNT-LHN) associated with membranes enriched in acidic phospholipids in a pH-dependent manner. Thus, in contrast to diphtheria toxin, the formation of a membrane-competent state of TeNT requires the membrane interface and is modulated by the bilayer composition. Channel formation is further enhanced by tethering of TeNT to the membrane through ganglioside co-receptors prior to acidification. Thus, TeNT channel formation can be resolved into two sequential steps: 1) interaction of the receptor binding domain (heavy chain receptor binding domain) with ganglioside co-receptors orients the translocation domain (heavy chain translocation domain) as the lumen of the endosome is acidified and 2) low pH, in conjunction with acidic lipids within the membrane drives the conformational changes in TeNT necessary for channel formation.

[Shiga toxin and tetanus toxin as a potential biologic weapon]. / Toksyna Shiga oraz toksyna tezcowa jako potencjalna bron.

Toxins produced by the bacteria are of particular interest as potential cargo combat possible for use in a terrorist attack or war. Shiga toxin is usually produced by shiga toxigenic strains of Escherichia coli (STEC - shigatoxigenic Escherichia coli). To infection occurs mostly after eating contaminated beef. Clinical syndromes associated with Shiga toxin diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS - hemolytic uremic syndrome) or thrombotic thrombocytopenic purpura. Treatment is symptomatic. In HUS, in which mortality during an epidemic reaches 20%, extending the kidney injury dialysis may be necessary. Exposure to tetanus toxin produced by Clostridium tetani, resulting in the most generalized tetanus, characterized by increased muscle tension and painful contractions of individual muscle groups. In the treatment beyond symptomatic behavior (among others spasticity medications, anticonvulsants, muscle relaxants) is used tetanus antitoxin and antibiotics (metronidazole choice). A common complication is acute respiratory failure - then it is necessary to implement mechanical ventilation.

Collaborative study for the characterisation of the BINACLE Assay for in vitro detection of tetanus toxicity in toxoids - Part 1.

For several decades the European Pharmacopoeia monographs Tetanus vaccine (adsorbed) (0452) and Tetanus vaccine for veterinary use (0697) required that Specific toxicity and Absence of toxin and irreversibility of the toxoidof each bulk of tetanus toxoids had to be tested by an in vivo toxicity test in guinea pigs before it could be included in vaccines for human or veterinary use. In line with the 3Rs concept of replacing, reducing and refining animal experiments, an in vitro method for the detection of active tetanus neurotoxin (TeNT) has been developed at the Paul-Ehrlich-Institut (PEI, Germany). This method, the so-called BINACLE (binding and cleavage) assay, uses the receptor-binding and proteolytic properties of TeNT for the specific detection of active toxin molecules. Successful in-house validation studies as well as a small-scale transferability study had demonstrated that this method may represent a suitable alternative to the compendial in vivo toxicity test. As a follow up, an international collaborative study aimed at verifying the suitability of the BINACLE assay as a potential alternative to the guinea pig toxicity test for tetanus toxoids was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) under the aegis of its Biological Standardisation Programme (BSP). Within the framework of this study, coded BSP136, a feasibility phase - also referred to as Phase 1 - was run to select and qualify critical study reagents and samples and to assess the performance of the BINACLE Standard Operating Procedure developed by the project leaders. Then the international collaborative study aimed at evaluating the BINACLE, referred to as BSP136 Phase 2, was started. A total of 19 international laboratories (comprising vaccine manufacturers as well as national control laboratories) were supplied with a detailed assay protocol, critical reagents required for the assay, three samples consisting of three different bulk tetanus toxoids donated by major European vaccine manufacturers and one international standard toxoid. Each of the participants was asked to perform three independent BINACLE assays following the provided protocol. The statistical analysis of the results showed that most of the participating laboratories were able to perform the BINACLE assay according to the provided protocol. However, the results obtained by the participants varied widely, and not all the laboratories were able to achieve a sensitive detection of active TeNT. Multiple factors may have contributed to the elevated variability of the BSP136 study results. From an analysis of these factors, strategies were developed to help increase the standardisation of the BINACLE assay and obtain more consistent results in a follow-up validation study, BSP 136 Phase 3 (Part 2), for which the experimental phase took place in 2023. The present manuscript summarises the outcome of Phases 1 and 2, which constitute Part 1 of the BSP136 project.

Collaborative study for the characterisation of the BINACLE Assay for in vitro detection of tetanus toxicity in toxoids - Part 2.

Tetanus vaccines for human and veterinary use are produced by formaldehyde-induced inactivation of tetanus neurotoxin (TeNT) purified from Clostridium tetani cultures. Due to the high morbidity caused by exposure to TeNT it is essential that the quality control of tetanus vaccines includes testing for absence of tetanus toxin as prescribed by European Pharmacopoeia monographs 0452 and 0697. Currently this test is carried out in guinea pigs for each bulk of tetanus toxoid. To test the applicability of the in vitro BINACLE ("binding and cleavage") assay as an alternative method for the quality control of tetanus vaccines, two collaborative studies were run by the European Directorate for the Quality of Medicines & HealthCare under the aegis of the Biological Standardisation Programme. The first collaborative study indicated that the method allows sensitive TeNT detection. However, a clear conclusion could not be drawn due to the high variability of the results. To address the variability, the protocol was optimised and further standardised for the second study. The study results demonstrated good assay precision, both with respect to repeatability and reproducibility. Importantly, the limit of detection was 0.11 ng/mL TeNT in five out of nine laboratories and 0.33 ng/mL in four out of nine laboratories, suggesting that the BINACLE assay can detect TeNT with similar sensitivity as in vivo toxicity tests and can thus be taken into consideration as an alternative method to the current compendial in vivo test.

Dissociation Between the Epileptogenic Lesion and Primary Seizure Onset Zone in the Tetanus Toxin Model of Temporal Lobe Epilepsy.

Despite extensive temporal lobe epilepsy (TLE) research, understanding the specific limbic structures' roles in seizures remains limited. This weakness can be attributed to the complex nature of TLE and the existence of various TLE subsyndromes, including non-lesional TLE. Conventional TLE models like kainate and pilocarpine hinder precise assessment of the role of individual limbic structures in TLE ictogenesis due to widespread limbic damage induced by the initial status epilepticus. In this study, we used a non-lesional TLE model characterized by the absence of initial status and cell damage to determine the spatiotemporal profile of seizure initiation and limbic structure recruitment in TLE. Epilepsy was induced by injecting a minute dose of tetanus toxin into the right dorsal hippocampus in seven animals. Following injection, animals were implanted with bipolar recording electrodes in the amygdala, dorsal hippocampus, ventral hippocampus, piriform, perirhinal, and entorhinal cortices of both hemispheres. The animals were video-EEG monitored for four weeks. In total, 140 seizures (20 seizures per animal) were analyzed. The average duration of each seizure was 53.2+/-3.9 s. Seizure could initiate in any limbic structure. Most seizures initiated in the ipsilateral (41 %) and contralateral (18 %) ventral hippocampi. These two structures displayed a significantly higher probability of seizure initiation than by chance. The involvement of limbic structures in seizure initiation varied between individual animals. Surprisingly, only 7 % of seizures initiated in the injected dorsal hippocampus. The limbic structure recruitment into the seizure activity wasn't random and displayed consistent patterns of early recruitment of hippocampi and entorhinal cortices. Although ventral hippocampus represented the primary seizure onset zone, the study demonstrated the involvement of multiple limbic structures in seizure initiation in a non-lesional TLE model. The study also revealed the dichotomy between the primary epileptogenic lesion and main seizure onset zones and points to the central role of ventral hippocampi in temporal lobe ictogenesis.

Dentate gyrus progenitor cell proliferation after the onset of spontaneous seizures in the tetanus toxin model of temporal lobe epilepsy.

Temporal lobe epilepsy alters adult neurogenesis. Existing experimental evidence is mainly from chronic models induced by an initial prolonged status epilepticus associated with substantial cell death. In these models, neurogenesis increases after status epilepticus. To test whether status epilepticus is necessary for this increase, we examined precursor cell proliferation and neurogenesis after the onset of spontaneous seizures in a model of temporal lobe epilepsy induced by unilateral intrahippocampal injection of tetanus toxin, which does not cause status or, in most cases, detectable neuronal loss. We found a 4.5 times increase in BrdU labeling (estimating precursor cells proliferating during the 2nd week after injection of toxin and surviving at least up to 7days) in dentate gyri of both injected and contralateral hippocampi of epileptic rats. Radiotelemetry revealed that the rats experienced 112±24 seizures, lasting 88±11s each, over a period of 8.6±1.3days from the first electrographic seizure. On the first day of seizures, their duration was a median of 103s, and the median interictal period was 23min, confirming the absence of experimentally defined status epilepticus. The total increase in cell proliferation/survival was due to significant population expansions of: radial glial-like precursor cells (type I; 7.2×), non-radial type II/III neural precursors in the dentate gyrus stem cell niche (5.6×), and doublecortin-expressing neuroblasts (5.1×). We conclude that repeated spontaneous brief temporal lobe seizures are sufficient to promote increased hippocampal neurogenesis in the absence of status epilepticus.

Time course and temperature dependence of the membrane translocation of tetanus and botulinum neurotoxins C and D in neurons.

Tetanus and botulinum neurotoxins act inside nerve terminals and, therefore, they have to translocate across a membrane to reach their targets. This translocation is driven by a pH gradient, acidic on the cis side and neutral on the cytosol. Recently, a protocol to induce translocation from the plasma membrane was established. Here, we have used this approach to study the temperature dependence and time course of the entry of the L chain of tetanus neurotoxin and of botulinum neurotoxins type C and D across the plasma membrane of cerebellar granular neurons. The time course of translocation of the L chain varies for the three neurotoxins, but it remains in the range of minutes at 37 °C, whilst it takes much longer at 20 °C. BoNT/C does not enter neurons at 20 °C. Translocation also depends on the dimension of the pH gradient. These data are discussed with respect to the contribution of the membrane translocation step to the total time to paralysis and to the low toxicity of these neurotoxins in cold-blood vertebrates.

[Preclinical studies of an adsorbed diphtheria-tetanus-pertussis vaccine (ADTP-vaccine) with acellular pertussis component].

AIM: Evaluate standardness of antigenic composition of pertussis component, completeness of sorption of pertussis, diphtheria and tetanus components, specific activity and safety of experimental series ofADTP-vaccine with acellular pertussis component (ADTaP-vaccine). MATERIALS AND METHODS: The content of separate antigens (pertussis toxin, filamentous hemagglutinin and agglutinogens 1, 2, 3) in samples of acellular pertussis component of ADTaP-vaccine and completeness of sorption of pertussis component of ADTaP-vaccine were evaluated by using enzyme immunoassay. Completeness of sorption of diphtheria and tetanus components were determined in flocculation reaction and antitoxin-binding reactions, respectively. Protective activity ofADTaP-vaccine was studied in model ofmeningoencephalitis development in mice infected with Bordetella pertussis (strain 18323) neurotropic virulent culture, protective activity oftetanus component - by survival of mice after administration of tetanus toxin, protective activity of diphtheria component - by survival of guinea pigs after administration of diphtheria toxin. Safety of preparations was evaluated in tests of acute and chronic toxicity with carrying out pathomorphologic studies including immature animals. RESULTS: All the studied experimental series ofADTaP-vaccine were standard by content of separate antigens of pertussis microbe. All the ADTaP-vaccine components were completely sorbed on aluminium hydroxide gel. By protective activity ADTaP preparations satisfied the WHO requirements. The preparations were non-toxic in acute and chronic toxicity and did not induce pathomorphologic changes including immature animals. CONCLUSION: Experimental samples of ADTaP-vaccine by specific activity and safety satisfied WHO requirements.

Gliosis in the mouse hippocampus without neuronal death after systemic administration of high dosage of tetanus toxin.

Tetanus toxin (TeT), an exotoxin, has been studied to cause tetanus in mammalian brains, and it can block the release of some neurotransmitters and affect seizure propagation. In the present study, we investigated neuronal damage/death and glial changes in the mouse hippocampus after systemic administration (intraperitoneal injection) of TeT 10 and 100 ng/kg. In both the 10 and 100 ng/kg TeT-treated groups, no neuronal death occurred in any subregions of the mouse hippocampus until 24 h post-treatment; however, there were changes in glia in the hippocampus depending on time course and dosage. The morphology of GFAP-immunoreactive astrocytes and Iba-1-immunoreactive microglia was apparently changed in the 100 ng/kg TeT treated-group compared to the 10 ng/kg TeT treated-group. In the 100 ng/kg TeT treated-group, they were increased in size and their immunoreactivity was distinctively increased from 12 h post-treatment. We also found that their protein levels were increased in the hippocampus at 12 h post-treatment of 100 ng/kg TeT. In conclusion, these results indicate that the systemic administration of 100 ng/kg TeT induced a distinctive microglia changes in the mouse hippocampus without any neuronal death/damage.

Tetanus neurotoxin-induced epilepsy in mouse visual cortex.

Tetanus neurotoxin (TeNT) is a metalloprotease that cleaves the synaptic protein VAMP/synaptobrevin, leading to focal epilepsy. Although this model is widely used in rats, the time course and spatial specificity of TeNT proteolytic action have not been precisely defined. Here we have studied the biochemical, electrographic, and anatomic characteristics of TeNT-induced epilepsy in mouse visual cortex (V1). We found that VAMP cleavage peaked at 10 days, was reduced at 21 days, and completely extinguished 45 days following TeNT delivery. VAMP proteolysis was restricted to the injected V1 and ipsilateral thalamus, whereas it was undetectable in other cortical areas. Electrographic epileptiform activity was evident both during and after the time window of TeNT effects, indicating development of chronic epilepsy. Anatomic analyses found no evidence for long-term tissue damage, such as neuronal loss or microglia activation. These data show that TeNT reliably induces nonlesional epilepsy in mouse cortex. Due to the excellent physiologic knowledge of the visual cortex and the availability of mouse transgenic strains, this model will be useful for examining the network and cellular alterations underlying hyperexcitability within an epileptic focus.

Focal neocortical epilepsy affects hippocampal volume, shape, and structural integrity: a longitudinal MRI and immunohistochemistry study in a rat model.

PURPOSE: Extratemporal epilepsy often coincides with cognitive decline, which may be associated with hippocampal dysfunction. Severe hippocampal sclerosis can be detected with conventional neuroimaging in some patients with chronic extrahippocampal epilepsy (so-called dual pathology). However, subtle structural hippocampal changes may already develop at a much earlier phase, and in a larger number of patients. Our goal was to longitudinally characterize the development of bilateral hippocampal pathology in an experimental neocortical focal epilepsy model. METHODS: Focal unilateral neocortical epilepsy was induced by microinjection of tetanus toxin in the primary motor cortex in adult male Sprague-Dawley rats. Another group of age-matched rats served as controls. In both groups, structural magnetic resonance imaging (MRI) was performed at 1, 3, 7, and 10 weeks of follow-up. Bilateral hippocampi were outlined and macroscopically analyzed using a state-of-the-art point-based morphometry model. Hippocampal microstructural changes at the end of follow-up, 10 weeks after epilepsy induction, were assessed with postmortem standard cresyl-violet, Fluoro-Jade, proteolipid protein 1, vimentin, glial fibrillary acidic protein, and ionized calcium binding adaptor molecule 1 stainings. KEY FINDINGS: All rats in the injected group developed seizures. The ipsilateral hippocampal volume was on average 8.76 (mean) ± 3.32% (standard deviation) smaller in the epileptic animals as compared to controls (p = 0.01) during the 10 weeks of follow-up. The contralateral hippocampus showed a similar reduction of 8.49 (mean) ± 3.27% (standard deviation) in total volume (p = 0.02). Clear hippocampal shape differences were found between the two groups. The most affected areas after epilepsy induction were the bilateral dorso-mediorostral, dorsolateral, and ventrolateral areas of the hippocampi. Normal developmental shape changes of the hippocampus, as detected in control rats, were largely absent in the ipsilateral hippocampus of epileptic rats. Quantitative histologic analysis revealed significant neuronal loss in the hippocampus, most pronounced in the hilar subregion, globally impaired myelination, reactive astrocytosis, and activated microglia. We found a weak but significant correlation between the number of neurons and hippocampal volume (r = 0.25, p = 0.0025). SIGNIFICANCE: We found evidence of hippocampal pathology in both hemispheres following experimental focal neocortical epilepsy. The observed development of bilateral hippocampal pathology, with onset in the early stages of focal neocortical epilepsy, may be a significant factor in comorbidities, such as cognitive dysfunction, found in patients with extratemporal localization-related epilepsy.

The C-terminal domain of the heavy chain of tetanus toxin prevents the oxidative and nitrosative stress induced by acute toxicity of 1-methyl-4-phenylpyridinium, a rat model of Parkinson's disease.

The recombinant carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) exerts neuroprotective and neurorestorative effects on the dopaminergic system of animal models of Parkinson's disease (PD). The present study aimed to determine the effect of the Hc-TeTx fragment on the markers of oxidative stress and nitrosative stress generated by the acute toxicity of 1-methyl-4-phenylpyridinium (MPP+). For this purpose, the Hc-TeTx fragment was administered once a day in three 20 µg/kg consecutive injections into the grastrocnemius muscle of the rats, with an intra-striatal unilateral injection of 1 µL of MPP+ [10 µg/mL] then administered in order to cause a dopaminergic lesion. The results obtained show that the rats treated with Hc-TeTx plus MPP+ presented an increase in the expression of tyrosine hydroxylase (TH), a significantly greater decrease in the levels of the markers of oxidative stress, nitrosative stress, and neurodegeneration than that observed for the group injured with only MPP+. Moreover, it was observed that total superoxide dismutase (SOD) and copper/zinc SOD activity increased with the administration of Hc-TeTx. Finally, immunoreactivity levels were observed to decrease for the levels of 3-nitrotyrosine and the glial fibrillary acidic protein in the ipsilateral striatum of the rats treated with Hc-TeTx plus MPP+, in contrast with those lesioned with MPP+ alone. Our results demonstrate that the recombinant Hc-TeTx fragment may be a potent antioxidant and, therefore, could be suggested as a therapeutic tool against the dopaminergic neuronal impairment observed in the early stages of PD.

Compositional optimization and safety assessment of a hydrogel patch as a transcutaneous immunization device.

The development of a simple, easy-to-use, and non-invasive vaccination system is in high demand. For transcutaneous immunization (TCI), we previously developed a hydrogel patch formulation that accelerates the penetration of an antigen (Ag) through the stratum corneum (SC) and effectively elicits Ag-specific immune responses. The present studies were performed to optimize the composition and assess the safety of the patch formulation. A hydrogel patch with a water content ratio of 5% more effectively induced an immune response compared to patches with a different composition, suggesting that the moisture content of the hydrogel patch formulation has optimal ratio for SC hydration to promote Ag penetration through the SC. TCI using a hydrogel patch induced few local and systemic adverse reactions. Based on these results, we are now advancing translational research to evaluate the safety and efficacy of our novel TCI system in humans.