Toward Evidence-Based Severity Assessment in Mouse Models with Repeated Seizures: (II.) Impact of Surgery and Intrahippocampal Kainate.

INTRODUCTION: Chronic epilepsy models require neurosurgical procedures including depth electrode implants. The intrahippocampal kainate model is a frequently used chronic paradigm, which is based on chemoconvulsant administration and status epilepticus induction during the surgical procedure. This experimental approach raises the question of the extent to which this approach affects postsurgical recovery. In addition to the short- and long-term impact of the surgical intervention, a potential impact of highly frequent electrographic seizure events needs to be considered in the context of severity assessment. METHODS: Various behavioral, biochemical, and telemetric parameters were analyzed in four experimental groups of mice: 1st naive, 2nd with transmitter implants, 3rd with transmitter and electrode implants, and 4th with transmitter implants, electrode implants, and kainate-induced status epilepticus. RESULTS: During the early postsurgical phase, transmitter implants caused a transient impact on Mouse Grimace scores and intragroup increase of fecal corticosterone metabolites. Additional craniotomy was associated with an influence on total heart rate variability and fecal corticosterone metabolites. Heart rate and Irwin score increases as well as a prolonged increase in Mouse Grimace scores pointed to an added burden related to the induction of a nonconvulsive status epilepticus. Data from the chronic phase argued against a relevant influence of frequent electrographic seizures on behavioral patterns, fecal corticosterone metabolites, heart rate, and its variability. However, Irwin scores indicated long-term changes in some animals with increased reactivity, body tone, and Straub tail. Interestingly, selected behavioral and telemetric data from the early post-status epilepticus phase correlated with the frequency of electrographic seizure events in the chronic phase. CONCLUSION: In conclusion, our findings argue against the pronounced impact of highly frequent electrographic seizures on the well-being of mice. However, an increased level of nervousness in a subgroup of animals should be considered for handling procedures and refinement measures. In the early postsurgical phase, several parameters indicate an influence of the interventions with evidence that the nonconvulsive status epilepticus can negatively affect the recovery. Thus, the development and validation of refinement efforts should focus on this experimental phase. Finally, the datasets suggest that simple readout parameters may predict the long-term consequences of the epileptogenic insult. Respective biomarker candidates require further validation in the follow-up studies in models with subgroups of animals with or without epilepsy development.

Brain expression profiles of two SCN1A antisense RNAs in children and adolescents with epilepsy.

Objective: Heterozygous mutations within the voltage-gated sodium channel α subunit (SCN1A) are responsible for the majority of cases of Dravet syndrome (DS), a severe developmental and epileptic encephalopathy. Development of novel therapeutic approaches is mandatory in order to directly target the molecular consequences of the genetic defect. The aim of the present study was to investigate whether cis-acting long non-coding RNAs (lncRNAs) of SCN1A are expressed in brain specimens of children and adolescent with epilepsy as these molecules comprise possible targets for precision-based therapy approaches. Methods: We investigated SCN1A mRNA expression and expression of two SCN1A related antisense RNAs in brain tissues in different age groups of pediatric non-Dravet patients who underwent surgery for drug resistant epilepsy. The effect of different antisense oligonucleotides (ASOs) directed against SCN1A specific antisense RNAs on SCN1A expression was tested. Results: The SCN1A related antisense RNAs SCN1A-dsAS (downstream antisense, RefSeq identifier: NR_110598) and SCN1A-usAS (upstream AS, SCN1A-AS, RefSeq identifier: NR_110260) were widely expressed in the brain of pediatric patients. Expression patterns revealed a negative correlation of SCN1A-dsAS and a positive correlation of lncRNA SCN1A-usAS with SCN1A mRNA expression. Transfection of SK-N-AS cells with an ASO targeted against SCN1A-dsAS was associated with a significant enhancement of SCN1A mRNA expression and reduction in SCN1A-dsAS transcripts. Conclusion: These findings support the role of SCN1A-dsAS in the suppression of SCN1A mRNA generation. Considering the haploinsufficiency in genetic SCN1A related DS, SCN1A-dsAS is an interesting target candidate for the development of ASOs (AntagoNATs) based precision medicine therapeutic approaches aiming to enhance SCN1A expression in DS.

Development of behavioral patterns in young C57BL/6J mice: a home cage-based study.

Evidence exists that behavioral patterns only stabilize once mice reach adulthood. Detailed information about the course of behavioral patterns is of particular relevance for neuroscientific research and for the assessment of cumulative severity in genetically modified mice. The analysis considered five age groups focusing on behavioral assessments in the animals' familiar home cage environment during the adolescence phase. We confirmed age- and sex-specific differences for several of the behavioral parameters and fecal corticosterone metabolites. Interestingly, an age-dependent decline in saccharin preference was detected in female mice. Regardless of sex, relevant levels of burrowing activity were only observed during later developmental phases. The development of nest complexity following the offer of new material was affected by age in female mice. In female and male mice, an age-dependency was evident for wheel running reaching a peak at P 50. A progressive increase with age was also observed for Open field activity. The data sets provide guidance for behavioral studies and for development of composite measure schemes for evidence-based severity assessment in young mice. Except for the burrowing test, the different behavioral tests can be applied in different age groups during post-weaning development. However, age- and sex-specific characteristics need to be considered.

The impact of tethered recording techniques on activity and sleep patterns in rats.

Electrophysiological recordings in animals constitute frequently applied techniques to study neuronal function. In this context, several authors described tethered recordings as a semi-restraint situation with negative implications for animal welfare and suggested radiotelemetric setups as a refinement measure. Thus, we here investigated the hypothesis that tethered recordings exert measurable effects on behavioral and sleep patterns in Sprague-Dawley rats. Animals were kept in monitoring glass cages either with or without a head connection to a recording cable. Saccharin preference, nest building, serum corticosterone and fecal corticosterone metabolite levels were in a comparable range in both groups. The proportion of vigilance states was not affected by the cable connection. Minor group differences were detected in bout lengths distributions, with a trend for longer NREM and WAKE stages in animals with a cable connection. However, a relevant effect was not further confirmed by an analysis of the number of sleep/wake and wake/sleep transitions. The analysis of activity levels did not reveal group differences. However, prolonged exposure to the tethered condition resulted in an intra-group increase of activity. In conclusion, the comparison between freely moving vs tethered rats did not reveal major group differences. Our findings indicate that telemetric recordings only offer small advantages vs cabled set ups, though this may differ in other experimental studies where for example anxiety- or drug-induced effects are analyzed.

Impact of Toll-like receptor 2 expression in renal allograft rejection.

BACKGROUND: An important role of TLR2 has been shown in various experimental models of renal ischaemia/reperfusion injury. To study the expression of TLR2 in renal allograft rejection systematically, we established an experimental rat transplantation model. METHODS: TLR2 expression was analysed in 99 human renal allograft biopsies, and in rat allografts at Day 6 and 28 after experimental renal transplantation. To discriminate whether regulation of TLR2 was following immunological processes after allogeneic transplantation or was a consequence from ischaemia/reperfusion injury, control animals subjected to syngeneic transplantation or to ischaemia/reperfusion damage were also investigated. RESULTS: TLR2 mRNA was significantly elevated in rat allografts with acute rejection on Day 6 and decreased spontaneously towards Day 28. TLR2 induction correlated with renal function and TLR2 excretion in the urine of transplanted rats. TLR2 staining was also significantly increased in human allografts with acute rejection. TLR2 protein could be localized in tubular epithelial cells and vascular endothelial cells, and in CD68- and CD4-positive infiltrating cells. CONCLUSIONS: TLR2 is markedly up-regulated in both experimental and human acute renal allograft rejection. Our data suggest a role for TLR2 during allogen-dependent graft damage after renal transplantation.

Single chain Fv antibodies directed against the 37 kDa/67 kDa laminin receptor as therapeutic tools in prion diseases.

Transmissible spongiform encephalopathies are a group of neurological disorders associated with the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(c). The 37 kDa/67 kDa laminin receptor (LRP/LR) has been identified as a prion receptor and several lines of evidence strongly suggest that this protein plays a role during prion pathogenesis. Here we report the selection of recombinant single chain antibodies (scFvs) directed against LRP from naïve and synthetic phage scFv libraries for therapeutic application. Western blotting and FACS analysis confirmed a specific LRP/LR recognition pattern of the two selected scFvs S18 and N3. Both scFvs specifically interfered with the PrP/LRP interaction in vitro. High yield production of the scFvs of approx. 1mg/l of culture medium was achieved in E. coli. Passive immunotransfer of the scFv S18 antibody reduced PrP(Sc) levels by approx. 40% in the spleen of scrapie infected C57BL/6 mice 90 days post scFv injection, suggesting that scFv S18 interferes with peripheral PrP(Sc) propagation, without a significant prolongation of incubation and survival times.

Assistive technology to promote leisure and constructive engagement by two boys emerged from a minimal conscious state.

BACKGROUND: Post-coma persons with multiple disabilities may represent a challenge to rehabilitation centers, due to their clinical conditions. Moreover, they can failed to engage adaptive responses aimed at the self-management of environmental stimuli. OBJECTIVES: To assess the impact and social rating of a new assistive technology set-up for promoting constructive engagement by two post-coma boys emerged from a minimal conscious state. METHOD: During baseline sessions, the participants were provided with a mouse to manage the computer system. During intervention phases, a new technology was implemented, allowing both participants to manage environmental stimuli with a microswitch instead of the mouse. Furthermore, a social validation assessment was carried out, involving students as raters. RESULTS: Data showed an increasing of constructive engagement by both participants during intervention phases. Sixty psychology students (social raters) favoured the new technology on a six items questionnaire (i.e. enjoyment, suitability, rehabilitation, independence, daily context and support). CONCLUSIONS: The new technology was suitable, affordable, effective and socially preferable.

Comprehensive morphometric analysis of mononuclear cell infiltration during experimental renal allograft rejection.

The role of specific subtypes of infiltrating cells in acute kidney allograft rejection is still not clear and was so far not examined by different analyzing methods under standardized conditions of an experimental kidney transplantation model. Immunohistochemical staining of CD3, CD20 and CD68 was performed in rat allografts, in syngeneically transplanted rats and in control rats with a test duration of 6 and 28 days. The detailed expression and localization of infiltrating cells were analyzed manually in different kidney compartments under light microscope and by the two different morphometric software programs. Data were correlated with the corresponding kidney function as well as with histopathological classification. The information provided by the morphometric software programs on the infiltration of the specific cell types after renal transplantation was in accordance with the manual analysis. Morphometric methods were solid to analyze reliably the induction of cellular infiltrates after renal transplantation. By manual analysis we could clearly demonstrate the detailed localization of the specific cell infiltrates in the different kidney compartments. Besides infiltration of CD3 and CD68 infiltrating cells, a robust infiltration of CD20 B-cells in allogeneically transplanted rats, even at early time points after transplantation was detected. Additionally an MHC class I expression could reliable be seen in allogeneically transplanted rats. The infiltration of B-cells and the reliable antigen presentation might act as a silent subclinical trigger for subsequent chronic rejection and premature graft loss.

Technology-assisted programmes to promote leisure engagement in persons with acquired brain injury and profound multiple disabilities: two case studies.

OBJECTIVE. To evaluate technology-assisted programmes for enabling a woman and a man with brain injury and profound multiple disabilities to acquire leisure engagement. METHOD. The technology for the woman (Study I) involved a portable computer with mouse, a Clicker 4 software package, a touch/pressure microswitch, and an interface to connect the Clicker with the microswitch. This technology allowed the woman to choose with a simple hand response among four stimulus categories (e.g., watching a film and interacting with others), each of which included several alternatives. The technology for the man (Study II) involved a computer-based choice system that allowed him to select preferred songs through a microswitch-aided finger-movement response. RESULTS. Data showed that the two participants learned to use the technology available and selected among the stimulus events thus reaching positive leisure engagement. CONCLUSION. Technology-assisted programmes may provide persons with acquired brain injury and multiple disabilities leisure engagement opportunities.

Communication opportunities via special messaging technology for two post-coma persons with multiple disabilities.

This study extended the assessment of a special messaging technology with two additional post-coma adults who had emerged from a minimally conscious state, but showed multiple disabilities including profound motor and communication impairments. For each participant, the study involved an ABAB design, in which the A represented baseline phases and the B represented intervention phases with the special messaging technology. The technology involved a net-book computer provided with specific software, a global system for mobile communication (GSM) modem, microswitches, and prerecorded verbal lists of persons' names and messages. Both participants learned to send out and receive (listen to) messages independently during the intervention, thus providing clear support for previous data in the area. They sent out means of about three and 17 messages and received means of about two and six messages per 20- and 30-min session, respectively. The positive impact of the technology was discussed in relation to previous data in this area and the possibility of helping post-coma persons with multiple disabilities engage in basic communication with distant partners.

Use of microswitch technology and a keyboard emulator to support literacy performance of persons with extensive neuro-motor disabilities.

OBJECTIVE: To assess the effectiveness and acceptability of microswitch technology and a keyboard emulator to enable three participants with extensive neuro-motor disabilities to write words. METHOD: In Study I, two participants triggered an automatic scanning keyboard and selected/wrote letters via a small sliding movement of their hand(s) activating a touch/pressure panel (microswitch). In Study II, a third participant used the sliding movement and panel and a vocalization response with a voice-detecting microswitch. The sliding movement allowed her to light up the keyboard and select the letters and the vocalization to perform the scanning. RESULTS: Participants showed a better performance (shorter writing time) or an equally effective but less tiring performance with the new microswitch technology and response(s). They also preferred using this technology, and social validation ratings favoured such technology over previous solutions. CONCLUSION: The aforementioned technology may be useful to enable persons with extensive neuro-motor disabilities to write successfully.

Scrapie-infected transgenic mice expressing a laminin receptor decoy mutant reveal a prolonged incubation time associated with low levels of PrPres.

The 37-kDa/67-kDa laminin receptor (LRP/LR) was identified as a cell surface receptor for prion proteins. The laminin receptor mutant LRP102-295::FLAG interfered with PrP(Sc) propagation in murine neuronal cells presumably acting as a decoy in a transdominant negative fashion by trapping PrP molecules in the extracellular matrix. Here, we generated hemizygous transgenic mice expressing LRP102-295::FLAG in the brain. Scrapie-infected transgenic mice exhibit a significantly prolonged incubation time in comparison to scrapie-infected wild-type (FVB) mice. At the terminal stage, transgenic mice revealed significantly reduced proteinase-K-resistant PrP levels by 71% compared to wild-type mice. Our results recommend the laminin receptor decoy mutant as an alternative therapeutic tool for treatment of transmissible spongiform encephalopathies.

Microinjection of lentiviral vectors expressing small interfering RNAs directed against laminin receptor precursor mRNA prolongs the pre-clinical phase in scrapie-infected mice.

We examined therapeutic in vitro and in vivo approaches using lentivirus-based packaging of small interfering RNAs (siRNAs) targeting the non-integrin laminin receptor mRNA for treatment and prevention of prion disorders. Transfection of N2aSc(+) cells with recombinant plasmids expressing three different siRNAs, significantly reduced both the LRP (laminin receptor precursor) and PrP(Sc) levels by approximately 40-60 %. Stereotactic intracerebral microinjection of recombinant lentiviral vectors LVsiRNA-LRP 7 and 9 into the cortex of C57BL/6 wild-type mice resulted in a significant reduction of the LR levels in the cortex 15 days post-injection by 62 and 82 %, respectively. Intracerebral RML inoculation of C57BL/6 mice after microinjection with recombinant lentiviral vector LVsiRNA-LRP 7 into the hippocampus resulted in a significant reduction of both LRP and PrP(Sc) levels by 36 and 41 %, respectively, concomitant with a significant prolongation of the pre-clinical phase. Lentiviral vectors expressing siRNAs targeting LRP mRNA represent a novel delivery system for the treatment of transmissible spongiform encephalopathies.

Persons with multiple disabilities accessing stimulation and requesting social contact via microswitch and VOCA devices: new research evaluation and social validation.

The first of these two studies assessed whether 11 participants with multiple disabilities of 5.3-18.2 (M=10.7) years of age would succeed in combining a microswitch for accessing preferred environmental stimuli and a Voice Output Communication Aid (VOCA) for requesting social contact. The second study conducted a social validation assessment of the aforementioned microswitch-VOCA combination. Data showed that all participants learned to use the microswitch and the VOCA. Moreover, the 10 participants, who received a 1-month post-intervention check, largely maintained their responding. The social validation assessment indicated that the raters (i.e., 110 university psychology students) favored the combination of microswitch and VOCA over the microswitch or the VOCA alone, and hypothetical combinations of microswitches or VOCAs.

Delivery of single-chain antibodies (scFvs) directed against the 37/67 kDa laminin receptor into mice via recombinant adeno-associated viral vectors for prion disease gene therapy.

The 37/67 kDa laminin receptor (LRP/LR) acts as a receptor for prions providing a promising target for the treatment of prion diseases. Recently, we selected anti-LRP/LR single-chain antibodies (scFvs) and proved a reduction of the peripheral PrP(Sc) propagation by passive immunotransfer into scrapie-infected mice. Here, we report the development of an in vivo gene delivery system based on adeno-associated virus (AAV) vectors expressing scFvs-S18 and -N3 directed against LRP/LR. Transduction of neuronal and non-neuronal cells with recombinant (r)AAV serotype 2 vectors encoding scFv-S18, -N3 and -C9 verified the efficient secretion of the antibodies. These vectors were administered via stereotactic intracerebral microinjection into the hippocampus of C57BL/6 mice, followed by intracerebral inoculation with 10 % RML at the same site 2 weeks post-injection of rAAV. After 90 days post-infection, scFv-S18 and -N3 expression resulted in the reduction of peripheral PrP(Sc) propagation by approximately 60 and 32 %, respectively, without a significant prolongation of incubation times and survival. Proof of rAAV vector DNA in spleen samples by real-time PCR strongly suggests a transport or trafficking of rAAV from the brain to the spleen, resulting in rAAV-mediated expression of scFv followed by reduced PrP(Sc) levels in the spleen most likely due to the blockage of the prion receptor LRP/LR by scFv.

Transcriptome analysis reveals altered cholesterol metabolism during the neurodegeneration in mouse scrapie model.

To identify the dynamic transcriptional alterations in CNS during the development of prion disease, brains of scrapie-infected mice and age-matched, mock-inoculated controls were analyzed immediately before inoculation and at different time points post-inoculation using Affymetrix microarray technique. A total of 449 probe sets, representing 430 genes, showed differential expression between scrapie- and mock-inoculated mice over the time course. These genes could be separated into two clusters according to expression patterns: the genes in cluster 1 demonstrated lower mRNA levels in scrapie-infected brains when compared with mock-inoculated brains, whereas genes in cluster 2 showed higher mRNA levels in scrapie-infected brains. Functional analysis of differentially expressed genes revealed the most severely affected biological process: cholesterol metabolism. The expression patterns of the cholesterol-related genes indicated an inhibited cholesterol synthesis in the diseased brains. Conspicuously, a number of cluster 1 genes, including some of cholesterol-related genes, showed not only decreasing mRNA levels in scrapie-infected brains but also increasing mRNA levels in mock-inoculated brains with increasing age. Quantitative RT-PCR analysis of some cholesterol-related genes in untreated mice suggested that changes of the examined genes observed in mock-inoculated brains are mainly age related. This finding indicated a link between age-related genes and scrapie-associated neurodegeneration.

Anti-LRP/LR antibody W3 hampers peripheral PrPSc propagation in scrapie infected mice.

We identified the 37kDa/67kDa laminin receptor (LRP/LR) as a cell surface receptor for the cellular prion protein (PrP(c)) and the infectious prion protein (PrP(Sc)). Recently, we showed that anti-LRP/LR antibody W3 cured scrapie infected N2a cells. Here, we demonstrate that W3 delivered by passive immunotransfer into C57BL/6 mice reduced the PrP(Sc) content in the spleen significantly by 66%, demonstrating an impairment of the peripheral PrP(Sc) propagation. In addition, we observed a 1.8-fold increase in survival of anti-LRP/LR antibody W3 treated mice (mean survival of 31 days) compared to preimmune serum treated control animals (mean survival of 17 days). We conclude that the significant effect of anti-LRP/LR antibody W3 on the reduction of peripheral PrP(Sc) propagation might be due to the blockage of the prion receptor LRP/LR which is required, as previously shown in vitro, for PrP(Sc) propagation in vivo.

Mutant presenilin 1 alters synaptic transmission in cultured hippocampal neurons.

Mutations in presenilins are the major cause of familial Alzheimer disease, but the precise pathogenic mechanism by which presenilin (PS) mutations cause synaptic dysfunction leading to memory loss and neurodegeneration remains unclear. Using autaptic hippocampal cultures from transgenic mice expressing human PS1 with the A246E mutation, we demonstrate that mutant PS1 significantly depressed the amplitude of evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptor-mediated synaptic currents. Analysis of the spontaneous miniature synaptic activity revealed a lower frequency of miniature currents but normal miniature amplitude. Both alterations could be rescued by the application of a gamma-secretase blocker. On the other hand, the application of synthetic soluble Abeta42 in wild-type neurons induced the PS1 mutant phenotype on synaptic strength. Together, these findings strongly suggest that the expression of mutant PS1 in cultured neurons depresses synaptic transmission by causing a physical reduction in the number of synapses. This hypothesis is consistent with morphometic and semiquantitative immunohistochemical analysis, revealing a decrease in synaptophysin-positive puncta in PS1 mutant hippocampal neurons.