Hind Limb Paralysis Research Articles

Excitation-contraction coupling inhibitors potentiate the actions of botulinum neurotoxin type A at the neuromuscular junction.

Botulinum neurotoxin type A1 (BoNT/A) is one of the most potent neurotoxins known. At the same time, it is also one of the safest therapeutic agents used for the treatment of several human disorders and in aesthetic medicine. Notwithstanding great effectiveness, strategies to accelerate the onset and prolong BoNT/A action would significantly ameliorate its pharmacological effects with beneficial outcomes for clinical use. Here, we combined BoNT/A with two fast-acting inhibitors of excitation-contraction coupling inhibitors (ECCI), either the μ-conotoxin CnIIIC or dantrolene, and tested the effect of their co-injection on a model of hind-limb paralysis in rodents using behavioural, biochemical, imaging and electrophysiological assays. The BoNT/A-ECCI combinations accelerated the onset of muscle relaxation. Surprisingly, they also potentiated the peak effect and extended the duration of the three BoNT/A commercial preparations OnabotulinumtoxinA, AbobotulinumtoxinA and IncobotulinumtoxinA. ECCI co-injection increased the number of BoNT/A molecules entering motoneuron terminals, which induced a faster and greater cleavage of SNAP-25 during the onset and peak phases, and prolonged the attenuation of nerve-muscle neurotransmission during the recovery phase. We estimate that ECCI co-injection yields a threefold potentiation in BoNT/A pharmacological activity. Overall, our results show that the pharmacological activity of BoNT/A can be combined and synergized with other bioactive molecules and uncover a novel strategy to enhance the neuromuscular effects of BoNT/A without altering the neurotoxin moiety or intrinsic activity, thus maintaining its exceptional safety profile.

Rabies in free-ranging capybaras (Hydrochoerus hydrochaeris) on Anchieta Island, Ubatuba, Brazil.

Between December 2019 and January 2020, three cases of rabies were reported in free-ranging capybaras on Anchieta Island, Ubatuba-SP, Brazil. This 8.28km² island is located 540m offshore from the mainland. Two of the capybaras exhibited signs of hindlimb paralysis, and one was found dead. Rabies was diagnosed using the direct fluorescent antibody test (dFAT), while RT-qPCR and phylogenetic analysis of nucleotide sequences confirmed the presence of the vampire bat rabies virus (RABV) strain. Although no visible bat bite marks were found on the capybaras, vampire bats are known to inhabit the island. Other wildlife tested negative for rabies during this period, and no further rabies outbreaks have been observed since. Environmental changes and human activities, such as the disturbance of bat roosting sites, may have contributed to the incident. The detection of rabies in capybaras suggests a potential spillover from a vampire bat reservoir. Further investigation is needed to determine whether capybaras act as dead-end hosts or play a role in maintaining the rabies transmission cycle.

Epidemiology of Canine Wei Syndrome and Its Hemorheology Characteristics.

Canine paraplegia is a common condition in small animal medicine, referred to as Wei Syndrome (WS) in Traditional Chinese Veterinary Medicine (TCVM). Common clinical manifestations encompass hind limb paralysis, motor dysfunction, muscle atrophy, and the absence of pain perception. WS is considered a difficult-to-treat disease in small animal practice. The objective of this study was to investigate the epidemiology of canine WS and the characteristics of hemorheology. A total of 53 dogs with WS and 53 healthy dogs were included in this study. A retrospective case-controlled study design was employed. Data regarding the gender, season of WS occurrence, breed, and age of dogs with WS, as well as hemorheology from dogs with WS and healthy dogs, were collected and analyzed using SPSS 27.0. The study findings revealed that male dogs were more susceptible to WS (77.36%, 41/53). WS cases occurred more frequently in Winter (33.96%, 18/53), and were commonly found in Poodle breeds (43.40%, 23/53). The most affected age of WS was between 3 and 6 years old (54.72%, 29/53). Except for plasma viscosity and fibrinogen, the hemorheology indices of canine WS were significantly higher than those of healthy dogs (p < 0.05), especially in male dogs, Poodles and Bulldogs, those between 3 to 10 years, and in Autumn and Winter. This study provides evidence that male Poodles and Bulldogs aged 3 to 6 years are more prone to developing WS, with Winter being the season of high disease incidence. Abnormal hemorheology is a characteristic feature in dogs with WS, which should be considered during the treatment of WS.

Passive bicycle training stimulates epiphyseal bone formation and restores bone integrity independent of locomotor recovery in a rat spinal cord injury model.

It is unknown whether activity-based physical therapy (ABPT) modalities that mobilize the paralyzed limbs improve bone integrity at the highly fracture-prone epiphyseal regions of the distal femur and proximal tibia following severe spinal cord injury (SCI). In this study, 4-mo-old skeletally mature littermate-matched male Sprague-Dawley rats received either SHAM surgery or severe contusion SCI. At 1 wk postsurgery, SCI rats were stratified to undergo no-ABPT, two 20-min bouts/day of quadrupedal bodyweight-supported treadmill training (qBWSTT), or hindlimb passive isokinetic bicycle (cycle) training, 5 days/wk for another 3 wk. We assessed locomotor recovery and plantar flexor muscle mass, tracked cancellous and cortical bone microstructure at the distal femoral and proximal tibial epiphyses using in vivo microcomputed tomography (microCT), and evaluated bone turnover at the tibial epiphysis with histomorphometry. All SCI animals displayed persistent hindlimb paralysis and pervasive muscle atrophy. Over the initial 2 wk, which included 1 wk of no exercise and 1 wk of ABPT acclimation, a similar magnitude of bone loss developed in all SCI groups. Thereafter, cancellous bone loss and cortical bone decrements increased in the SCI no-ABPT group. qBWSTT attenuated this trabecular bone loss but did not prevent the ongoing cortical bone deficits. In comparison, twice-daily cycle training increased the number and activity of osteoblasts versus other SCI groups and restored all bone microstructural parameters to SHAM levels at both epiphyseal sites. These data indicate that a novel passive isokinetic cycle training regimen reversed cancellous and cortical bone deterioration at key epiphyseal sites after experimental SCI via osteoblast-mediated bone anabolic mechanisms, independent of locomotor recovery or increased muscle mass.NEW & NOTEWORTHY This study was the first to assess how quadrupedal bodyweight-supported treadmill training or passive isokinetic bicycle (cycle) training impacts bone recovery at the distal femoral and proximal tibial epiphyses in a rat model of severe contusion spinal cord injury. Our results demonstrate that passive isokinetic cycle training completely restored cancellous and cortical bone microstructural parameters at these sites via osteoblast-mediated bone anabolic actions, independent of locomotor recovery or increased plantar flexor muscle mass.

Biochemical Characteristics of Pharmacologically Induced Hind Limb Paralysis in CD-1 Mice

A model of pharmacological paralysis in the hind limbs of CD-1 mice was introduced. In the initial phase (before paralysis), the activity of MAO-A, a key enzyme of neuroamine metabolism, was inhibited, leading to increased levels of steroid hormones and prolactin, as well as to a decrease in hepatic CYP3A4 and CYP2D6 activities and astrocytic S-100 protein secretion into the blood serum. In the second phase (paralysis manifestation), the mice exhibited hind limb paralysis development, accumulation of cortisol granules, destruction of capillaries, and aggregation of deformed red blood cells in the cerebral cortex and hippocampal regions.

Transcriptomics reveals transient and dynamic muscle fibrosis and atrophy differences following spinal cord injury in rats.

The rate and magnitude of skeletal muscle wasting after severe spinal cord injury (SCI) exceeds most other disuse conditions. Assessing the time course of molecular changes can provide insight into the progression of muscle wasting post-SCI. The goals of this study were (1) to identify potential targets that may prevent the pathologic features of SCI in soleus muscles and (2) to establish therapeutic windows for treating these pathologic changes. Four-month-old Sprague-Dawley male rats received T9 laminectomy (SHAM surgery) or severe contusion SCI. Hindlimb locomotor function was assessed weekly, with soleus muscles obtained 1week, 2weeks, 1month and 3months post-surgery (n=6-7 per group per timepoint). RNA was extracted from muscles for bulk RNA-sequencing analysis (n=3-5 per group per timepoint). Differentially expressed genes (DEGs) were evaluated between age-matched SHAM and SCI animals. Myofiber size, muscle fibre type and fibrosis were assessed on contralateral muscles. SCI produced immediate and persistent hindlimb paralysis, with Basso-Beattie-Bresnahan locomotor scores remaining below 7 throughout the study, contributing to a progressive 25-50% lower soleus mass and myofiber atrophy versus SHAM (P<0.05 at all timepoints). Transcriptional comparisons of SCI versus SHAM resulted in 184 DEGs (1week), 436 DEGs (2weeks), 133 DEGs (1month) and 1200 DEGs (3months). Upregulated atrophy-related genes included those associated with cell senescence, nuclear factor kappa B, ubiquitin proteasome and unfolded protein response pathways, along with upregulated genes that negatively influence muscle growth through the transforming growth factor beta pathway and inhibition of insulin-like growth factor-I/Akt/mechanistic target of rapamycin and p38/mitogen-activated protein kinase signalling. Genes associated with extracellular matrix (ECM), including collagens, collagen crosslinkers, proteoglycans and those regulating ECM integrity, were enriched within upregulated DEGs at 1week but subsequently downregulated at 2weeks and 3months and were accompanied by >50% higher ECM areas and hydroxyproline levels in SCI muscles (P<0.05). Myofiber remodelling genes were enriched in upregulated DEGs at 2weeks and 1month and were downregulated at 3months. Genes that regulate neuromuscular junction remodelling were evident in muscles post-SCI, along with slow-to-fast fibre-type shifts: 1 week and 2 weeks SCI muscles were composed of 90% myosin heavy chain (MHC) type I fibres, which decreased to only 16% at 3months and were accompanied by 50% fibres containing MHC IIX (P<0.05). Metabolism genes were enriched in upregulated DEGs at 1month and were further enriched at 3months. Our results substantiate many known pathologic features of SCI-induced wasting in rat skeletal muscle and identify a progressive and dynamic transcriptional landscape within the post-SCI soleus. Future studies are warranted to consider these therapeutic treatment windows when countering SCI muscle pathology.

337 Cellular senescence contributes to inflammation and disease progression in an animal model of multiple sclerosis

OBJECTIVES/GOALS: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. MS affects more than two million people worldwide, resulting in physical disability, cognitive impairment, and decreased quality of life. We are investigating the role of senescent cells, a hallmark of aging, in inflammation and disease progression in MS. METHODS/STUDY POPULATION: Female mice on a C57BL/6 background were subjected to the Hooke Laboratory EK-2110 experimental autoimmune encephalomyelitis protocol (EAE; n=10) to induce hind limb paralysis, and matching control mice received no injections (naīve; n=10). Immunofluorescent staining was used to visualize senescence cells and their localization in spinal cord tissue sections from naīve and EAE mice based on antibody detection of cell surface or intracellular proteins. Tissue sections from each group were analyzed in duplicates for each antibody (n=3-4/group). Flow cytometry was performed to immunophenotype the senescence cells using conjugated fluorescent antibodies to cell surface or intracellular proteins (n=5/group). RESULTS/ANTICIPATED RESULTS: Immunostaining demonstrated an increase in the cell senescence markers p16 (15-fold; unpaired t-test, p=0.01; n=3) and p21 (15-fold; unpaired t-test, p=0.003; n=3) in the spinal cord of EAE mice compared to naīve mice. Next, we showed that p16+ and p21+ cells increase with disease progression in the meninges adjacent to the ventral demyelinating lesions (one-way ANOVA, p=0.0009; n=3/group). We further found that 30±9.7% of M2 macrophages, a subset of myeloid cells, express p21 in the spinal cord of EAE mice by using flow cytometry analysis compared to only 5±1.6% in naīve mice (unpaired t-test, p=0.002; n=5/group). DISCUSSION/SIGNIFICANCE: Our findings demonstrate that senescent cells accumulate in the meningeal compartment following EAE induction, suggesting that decreasing senescent cell burden is a promising avenue in delaying disease progression and preventing neuroinflammation in MS.

TMEM106B coding variant is protective and deletion detrimental in a mouse model of tauopathy.

TMEM106B is a risk modifier of multiple neurological conditions, where a single coding variant and multiple non-coding SNPs influence the balance between susceptibility and resilience. Two key questions that emerge from past work are whether the lone T185S coding variant contributes to protection, and if the presence of TMEM106B is helpful or harmful in the context of disease. Here, we address both questions while expanding the scope of TMEM106B study from TDP-43 to models of tauopathy. We generated knockout mice with constitutive deletion of TMEM106B, alongside knock-in mice encoding the T186S knock-in mutation (equivalent to the human T185S variant), and crossed both with a P301S transgenic tau model to study how these manipulations impacted disease phenotypes. We found that TMEM106B deletion accelerated cognitive decline, hind limb paralysis, tau pathology, and neurodegeneration. TMEM106B deletion also increased transcriptional correlation with human AD and the functional pathways enriched in KO:tau mice aligned with those of AD. In contrast, the coding variant protected against tau-associated cognitive decline, synaptic impairment, neurodegeneration, and paralysis without affecting tau pathology. Our findings reveal that TMEM106B is a critical safeguard against tau aggregation, and that loss of this protein has a profound effect on sequelae of tauopathy. Our study further demonstrates that the coding variant is functionally relevant and contributes to neuroprotection downstream of tau pathology to preserve cognitive function.

Spinal lymphoma in a goat.

A 3-year-old Pygmy Wether was presented for chronic hindlimb paralysis. A neurological exam revealed nonambulatory paraplegia with absent deep pain nociception, lack of hindlimb withdrawal reflexes, and paraspinal pain on palpation with T3 to L3 neurolocalization. MRI of the lumbar spine revealed an extensive, dorsal to dorsolateral, severely compressive, heterogeneously contrast-enhancing extradural lesion of the lumbar spine with intervertebral foraminal extension into the surrounding paraspinal musculature. Vertebral bone marrow involvement was also noted in the L5 and L6 vertebrae. A diagnosis of lymphoma was obtained after cytological sampling. This is the first case report describing specific MRI findings (signal characteristics, enhancement pattern, and perilesional changes) in a goat with paraspinal lymphoma.

Eugenol ameliorates uveitis in mice with experimental autoimmune encephalomyelitis through the suppression of key inflammatory genes

ABSTRACT Visual impairment associated with uveitis is among the potential complications in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Bioinformatics analyses have shown that some hub genes are closely associated with the molecular mechanisms underlying uveitis in EAE. This study evaluated whether 4-allyl-2-methoxyphenol (eugenol) can mitigate the pathogenesis of uveitis in EAE through the interruption of key uveitogenic gene expression. Myelin oligodendrocyte glycoprotein35–55 (MOG) peptide-immunized C57BL/6 mice were injected intraperitoneally with eugenol. The eyeballs and spinal cords of EAE mice with or without eugenol treatment were collected simultaneously and immunohistochemical and molecular biological analyses were conducted. Eugenol treatment significantly ameliorated hindlimb paralysis. Ionized calcium-binding adapter molecule 1 (Iba-1) immunohistochemistry showed that the inflammatory response was significantly reduced in the uvea of eugenol-treated EAE mice compared with vehicle-treated controls. Eugenol also significantly reduced the expression of key uveitogenic genes including C1qb and Tyrobp. The suppressive effect of eugenol on inflammation was also observed in the spinal cord, as determined by the suppression of Iba-1-positive microglial cells. Together, these results suggest that the ameliorative effect of eugenol against EAE uveitis is associated with the suppression of key proinflammatory genes, which may represent targets for the treatment of uveitis.

Evaluation of the antinociceptive effect of lidocaine- tramadol and lidocaine- medetomidine lumbosacral epidural anesthesia: A cross-over comparative study in goats

Small ruminants have a docile temperament and are typically operated under local or regional analgesia. In goats, lumbosacral anesthesia is the most commonly used regional anesthesia. This investigation aimed to evaluate the epidural anesthetic effects of lidocaine-medetomidine (LID-MED) and lidocaine-tramadol (LID-TRM) combinations in relation to cardiopulmonary effects. An experiment using a cross-over design was conducted on eight goats. The first group (LID-MED) was injected with lidocaine hydrochloride and medetomidine hydrochloride. The second group (LID-TRM) was injected with lidocaine hydrochloride and tramadol hydrochloride. The onset of analgesia, recumbency time, and standing time were recorded once, while scores were recorded periodically. Locomotor and anti-nociception scores were evaluated at baseline, 5, 10, 15, 30, 60, 90, and 120 minutes (min) post-anesthesia. Similarly, cardiorespiratory values were also recorded at the same intervals in each group. In the LID-MED receiving group, analgesia and recumbency onset were earlier, with a longer recumbency period. The LID-MED group showed a significant loss of sensation in all examined regions. The locomotor score revealed hind limb paralysis for 90 min in the LID-TRM group, while it continued for 120 min in the LID-MED group. In both LID-TRM and LID-MED groups, there was significant hypothermia; however, bradycardia was noticed in the LID-MED group from 5 min post-injection. Respiratory depression was also detected in the LID-MED group. The study revealed that lumbosacral epidural anesthesia using LID-TRM co-infusion produces reasonable and short duration (60 min) analgesia. In contrast, epidural lumbosacral injection of LID-MED co-infusion produces a longer duration of analgesia and recumbency.

Whole-Genome Sequence and Pathogenicity Analysis of Providencia Heimbachae Causing Diarrhea in Weaned Piglets.

Providencia heimbachae was previously identified in piglets with post-weaned diarrhea and associated with hindlimb paralysis. However, the pathogenic mechanisms and virulence factors of P. heimbachae are not fully known. Whole-genome sequence analysis will be helpful to extend our understanding of the characterization of P. heimbachae at a genomic level. In this study, we sequenced the whole genome of P. heimbachae for the first time using PacBio RS II sequencers and assembled de novo through hierarchical genome assembly process (HGAP). Furthermore, we performed further genome annotation. The genome of P. heimbachae 99101 consists of a circular chromosome (4,262,828bp) and a circular plasmid (231,957bp) with G + C contents of 40.43 and 47.16%, respectively. Genome-wide sequence analysis yielded a total of 286 predicted virulence factors, 178 resistance genes, 17 chaperone protein manipulators of fimbriae, 47 genes involved in the encoding of flagellin, 12 cell membrane-associated virulence genes, 18 Enterobacteriaceae common antigens, etc. Based on genome analysis, we preliminarily confirmed through animal experiments that the capsule was the virulence factor of P. heimbachae causing hindlimb paralysis in animals. Our study provides a genetic basis for further elucidation of the characteristics and functional mechanisms of P. heimbachae as a conditionally pathogenic bacterium, as well as a direction for research into the mechanism of action of P. heimbachae infecting humans, extending knowledge of P. heimbachae as an important zoonotic pathogen.

Adverse effect of botulinum toxin-A injections on mandibular bone: A systematic review and meta-analysis.

Botulinum toxin-A (BTX) is a potent neurotoxin that is emerging in the scope of dental practice for its ability to temporarily paralyse musculature and reduce hyperfunction. This may be desirable in diseases/disorders associated with hyperactive muscles such as the muscles of mastication, most implicated in painful temporomandibular disorders (TMDs). The use of BTX extends beyond its indications with off-label use in TMD's and other conditions, while potential adverse effects remain understudied. BTX is well-established hindlimb paralysis model in animals leading to significant bone loss with underlying mechanisms remaining unclear. The objective of this study is to systematically review the literature for articles investigating changes in mandibular bone following BTX injections and meta-analyse available data on reported bone outcomes. Comprehensive search of Medline, Embase and Web of Science retrieved 934 articles. Following the screening process, 36 articles in animals and humans were included for quantitative synthesis. Articles in human individuals (6) and three different animal species (14) presented mandibular bone outcomes that were included in the meta-analysis. The masseter and temporalis muscles were frequently injected across all species. In humans, we observe a decrease of about 6% in cortical thickness of mandibular regions following BTX injection with no evident changes in either volume or density of bone structures. In animals, bone loss in the condylar region is significantly high in both cortical and trabecular compartments. Our analysis supports the concept of BTX-induced bone-loss model in animal mandibles. Further, bone loss might be confined to the cortical compartments in humans. Most studies did not address the reality of repeated injections and excessive dosing, which occur due to the reversible action of BTX. More rigorous trials are needed to draw a full picture of potential long-term adverse effects on bone.

G9a inactivation in progenitor cells with Isl1-Cre with reduced recombinase activity models aspects of Dandy-Walker complex.

G9a, also known as EHMT2, is essential for embryogenesis and has specific functions in multiple developmental processes. G9a inactivation affects development of the nervous system, which is formed with contribution of descendants of progenitor cells expressing the transcription factor Isl1. However, the function of G9a in Isl1-expressing progenitors is unknown. Here, we show that G9a is required for proper development of multiple structures formed with contribution of Isl1-expressing progenitors. A Cre-dependent GFP reporter revealed that the recombinase activity of the Isl1-Cre used in this study to inactivate G9a was reduced to a subset of Isl1-expressing progenitor cells. G9a mutants reached endpoint by 7 weeks of age with cardiac hypertrophy, hydrocephalus, underdeveloped cerebellum and hind limb paralysis, modeling aspects of Dandy-Walker complex. Moreover, neuroepithelium of the lateral ventricle derived from Isl1-expressing progenitors was thinner and disorganized, potentially compromising cerebrospinal fluid dynamics in G9a mutants. Micro-computed tomography after iodine staining revealed increased volume of the heart, eye lens and brain structures in G9a mutant fetuses. Thus, altered development of descendants of the second heart field and the neural crest could contribute to multicomponent malformation like Dandy-Walker.

Lack of antiviral activity of ivermectin against foot-and-mouth disease virus serotype O in BALB/c mice

Ivermectin is an FDA approved drug and showed in vitro antiviral activity against different serotypes of Foot-and-mouth disease virus (FMDV). We here assessed the effect of ivermectin in 12 day old female BALB/c mice infected with 50LD50 FMDV serotype O intraperitoneally. Initially FMDV was adopted on 3-day old BALB/c mice by blind passages. After successful adaptation of virus mice showed hind limb paralysis. Mice were divided in 6 different groups and each group has 6 mice. Ivermectin was given at clinically prescribed dose of 500 μg/kg subcutaneously at different time interval. Ivermectin was given at 0 h post infection (hpi) and 12 hpi. Moreover we compared commercially available ivermectin with purified ivermectin preparation in sterilized DMSO. Viral load was evaluated through RT-qPCR and ELISA in different groups. Results showed that positive control and negative control has CT-value 26.28 and 38 respectively. Treated groups at 0hpi, 12hpi, purified ivermectin and pre-post treatment group has CT values 24.89, 29.44, 27.26 and 26.69 respectively that showed there was no significant reduction in virus load in treated groups as compare to positive control. In histopathology of lung tissue perialveolar capillaries were congested and alveoli were altelactic. Some emphysema was seen in alveoli and mild thickening in the alveolar wall was observed. In the alveolar epithelium mononuclear cells infiltration was seen. There was discoloration haemorrhages and enlargement of heart. Degeneration, fragmentation and loss of sarcoplasm were seen in the cardiac muscle fibers. Above results showed that ivermectin did not lessen lung and heart viral load. This study contributes that ivermectin does not have a significant antiviral effect when used in mice against FMDV serotype O, according to a growing body of research.

Abaloparatide prevents immobilization-induced cortical but not trabecular bone loss after spinal cord injury.

Spinal cord injury (SCI) causes severe and resistant sublesional disuse bone loss. Abaloparatide, a modified parathyroid hormone related peptide, is an FDA approved drug for treatment of severe osteoporosis with potent anabolic activity. The effects of abaloparatide on SCI-induced bone loss remain undefined. Thus, female mice underwent sham or severe contusion thoracic SCI causing hindlimb paralysis. Mice then received subcutaneous injection of vehicle or 20 μg/kg/day abaloparatide for 35 days. Micro-computed tomography (micro-CT) analysis of the distal and midshaft femoral regions of the SCI-vehicle mice revealed reduced trabecular fractional bone volume (56%), thickness (75%), and cortical thickness (80%) compared to sham-vehicle controls. Treatment with abaloparatide did not prevent SCI-induced changes in trabecular or cortical bone. However, histomorphometry evaluation of the SCI-abaloparatide mice demonstrated that abaloparatide treatment increased osteoblast (241%) and osteoclast (247%) numbers and the mineral apposition rate (131%) compared to SCI-vehicle animals. In another independent experiment, treatment with 80 μg/kg/day abaloparatide significantly attenuated SCI-induced loss in cortical bone thickness (93%) when compared to SCI-vehicle mice (79%) but did not prevent SCI-induced trabecular bone loss or elevation in cortical porosity. Biochemical analysis of the bone marrow supernatants of the femurs showed that SCI-abaloparatide animals had 2.3-fold increase in procollagen type I N-terminal propeptide, a bone formation marker than SCI-vehicle animals. SCI groups had 70% higher levels of cross-linked C-telopeptide of type I collagen, a bone resorption marker, than sham-vehicle mice. These findings suggest that abaloparatide protects the cortical bone against the deleterious effects of SCI by promoting bone formation.

A novel tumor-bearing humanized mouse model for evaluating the efficacy and predictive safety of bispecific T cell engager

Abstract Bispecific T cell engager (BiTE) is one of the most promising therapeutic approaches for treatment of various malignancies. Humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs) have currently become the preclinical platform for evaluating the effectiveness of various cancer immunotherapies including BiTE. However, the wide application of this humanized animal model is limited by a strong xenoreaction of human T cells against the mouse tissues. In this study, we used humanized murine major histocompatibility class I and class II double knock-out NSG mice (referred to DKO) to evaluate the antitumor effect of BiTE. The DKO mice were implanted with human pre-B acute lymphoblastic leukemia cell line (Nalm6). Five days later, the mice were reconstituted with PBMCs. Following CD19xCD3 BiTE treatment whole-body tumor burden was quantified by a in vivo imaging system. Our data demonstrated that the mice developed consistent clinical signs of leukemia-related body weight loss and hind-limb paralysis within 4 weeks. With serial dose of CD19xCD3 BiTE treatment, the tumor cell growing was significantly suppressed. A lower incidence of hind-limb paralysis with prolonged survival were observed in BiTE-treated mice compared with controls. In addition, after treatment with CD19xCD3 BiTE, the humanized mice exhibited elevated serum levels of human IFN-γ, TNF-a, IL-6, IL-10, IL-2, and IL-4. The cytokine production was dose-dependent and associated with the increase of CD19xCD3 BiTE effectiveness. This tumor-bearing humanized model provides a promising preclinical platform for evaluating the efficacy, toxic cytokine release, and dosing requirements of any T cell-based immunotherapies for cancer treatment.

B cells Regulation by Interferon-gamma in a Neuromyelitis Optica Spectrum Disorder animal model

Abstract Neuromyelitis Optica Spectrum Disorder (NMOSD) is an autoimmune disease of the central nervous system (CNS) mediated by Th17 and auto-antibody response against the water channel protein Aquaporin-4 (AQP4). In NMOSD, B cells plays a central role regulating autoreactive T and antibody secreting cells, and the use of anti-B cells therapy is a common practice for patients. Despite this, the mechanisms of B cells response against AQP4 are poorly understood. We have demonstrated that the immune tolerance against the immunodominant AQP4 201–220peptide in mice is maintained by IFN-g, controlling disease incidence, severity, and chronicity. Actively immunization of C57BL/6 (WT) treated with neutralizing anti-IFN-g antibodies, IFN-g knockout (IFNGKO) and IFN-g receptor KO (IFNRKO) mice was induced using the AQP4 201–220peptide, showing an ascendant paralysis starting from the tail to complete hind limb paralysis. CNS histological analysis showed an increased presence of T and B cells, and the complement system in lesion sites, associated with CNS damage. CNS Th17 profile was also observed in the anti-IFN-g treated group. Conversely, IFN-g treatment in IFNGKO mice decrease disease severity with a reduction of CNS CD4 +and B cells. Remarkably, IFN-g regulates the IL-6 expression by B cells in an antigen specific fashion. B-cell depletion significantly reduced the disease incidence, T cells CNS infiltration and anti-AQP4 201–220serum antibodies. Finally, using anti-IL-6R treatment, we showed that IL-6 signaling is necessary for the B cell activation and their induction of Th17 cells. In conclusion, AQP4 201–220tolerance is strictly regulated by IFN-g and its signaling, restraining B cells pathogenicity through IL-6 signaling modulation. This work was supported by National Institutes of Health grants R21AI151438 and R01 NS099334

Abstract 20: In vitro and in vivo investigation of tumor treating fields for treatment of spinal metastasis

Abstract Introduction: Tumor treating fields (TTF) are used for treatment of glioblastoma. Its utilization for spinal metastasis has never been described. We investigate the anti-mitotic effect of TTF to inhibit the growth of human cancer cells in vitro and in vivo models of spinal metastasis. Methods: In vitro: human derived osteosarcoma (Krib-luc-1) and lung adenocarcinoma (A549-luc) cells were seeded in 5 × 3mm human demineralized bone grafts kept in culture while treated with 0kHz (control) or 150kHZ of TTF for 14 days. Bioluminescence images (BLI) were obtained at baseline and 14 days. In vivo: Krib-Luc-1 cells were surgically implanted via transperitoneal approach to L3 vertebra in nude mice. Animals were treated with 0kHz or 150kHz for 30 days or until death. BLI were obtained at day 0 and every 7 days. MRI was obtained at day 22. Histological analysis was performed at the end of the experiment. Control and TTF-treated groups were compared with two-tailed t-tests. Results: In vitro: BLI of the control groups demonstrated 5-fold and 34-fold higher tumor signal (photons/sec) than corresponding 150 kHz TTF treatment groups at 14 days (A549 p=0.0289; KRIB p=0.00895). In vivo: All mice receiving 0 kHz treatment displayed hind limb paralysis by day 30, while mice treated with 150 kHz TTF displayed tail drop or no signs of paralysis. MRI and histology confirmed smaller tumors corroborating to BLI (3.67-fold change in photons/sec) (p=0.0212) when the 150 kHz TTF treatment group was compared to controls. Conclusions: Application of TTF at 150kHz reduced cellular proliferation in both in vitro and in vivo models of spinal metastasis. This is the first report of utilization of TTF for treatment of bony tumors and represents the first evidence that TTF could become a revolutionary therapy for spinal metastasis. Citation Format: Claudio Esteves Tatsui, Daniel Ledbetter, Christopher Alvarez-Breckenridge. In vitro and in vivo investigation of tumor treating fields for treatment of spinal metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 20.

Effect of Nrf2 loss on senescence and cognition of tau-based P301S mice.

Cellular senescence may contribute to chronic inflammation involved in the progression of age-related diseases such as Alzheimer's disease (AD), and its removal prevents cognitive impairment in a model of tauopathy. Nrf2, the major transcription factor for damage response pathways and regulators of inflammation, declines with age. Our previous work showed that silencing Nrf2 gives rise to premature senescence in cells and mice. Others have shown that Nrf2 ablation can exacerbate cognitive phenotypes of some AD models. In this study, we aimed to understand the relationship between Nrf2 elimination, senescence, and cognitive impairment in AD, by generating a mouse model expressing a mutant human tau transgene in an Nrf2 knockout (Nrf2KO) background. We assessed senescent cell burden and cognitive decline of P301S mice in the presence and absence of Nrf2. Lastly, we administered 4.5-month-long treatments with two senotherapeutic drugs to analyze their potential to prevent senescent cell burden and cognitive decline: the senolytic drugs dasatinib and quercetin (DQ) and the senomorphic drug rapamycin. Nrf2 loss accelerated the onset of hind-limb paralysis in P301S mice. At 8.5months of age, P301S mice did not exhibit memory deficits, while P301S mice without Nrf2 were significantly impaired. However, markers of senescence were not elevated by Nrf2 ablation in any of tissues that we examined. Neither drug treatment improved cognitive performance, nor did it reduce expression of senescence markers in brains of P301S mice. Contrarily, rapamycin treatment at the doses used delayed spatial learning and led to a modest decrease in spatial memory. Taken together, our data suggests that the emergence of senescence may be causally associated with onset of cognitive decline in the P301S model, indicate that Nrf2 protects brain function in a model of AD through mechanisms that may include, but do not require the inhibition of senescence, and suggest possible limitations for DQ and rapamycin as therapies for AD.