Pig Spinal Cord Research Articles

Spinal cord lesion MRI and behavioral outcomes in a miniature pig model of spinal cord injury: exploring preclinical potential through an ad hoc comparison with human SCI.

prospective case series of Yucatan miniature pig spinal cord contusion injury model with comparison to human cases of spinal cord injury (SCI). to describe magnetic resonance imaging (MRI) measures of spinal cord lesion severity along with estimates of lateral corticospinal tracts spared neural tissue in both a less severe and more severe contusion SCI model, as well as to describe their corresponding behavioral outcome changes. University laboratory setting. Following a more severe and less severe SCI, each pig underwent spinal cord MRI to measure lesion characteristics, along with locomotor and urodynamics outcomes testing. In the pig with more severe SCI, locomotor and urodynamic outcomes were poor, and both the spinal cord lesion volume and damage estimates to the lateral corticospinal tracts were large. Conversely, in the pig with less severe SCI, locomotor and urodynamic outcomes were favorable, with the spinal cord lesion volume and damage estimates to the lateral corticospinal tracts being less pronounced. For two human cases matched on estimates of damage to the lateral corticospinal tract regions, the clinical presentations were similar to the pig outcomes, with more limited mobility and more limited bladder functional independence in the more severe case. Our initial findings contribute valuable insights to the emergent field of MRI-based evaluation of spinal cord lesions in pig models, offering a promising avenue for understanding and potentially improving outcomes in spinal cord injuries.

Real-time in vivo thoracic spinal glutamate sensing during myocardial ischemia.

In the spinal cord, glutamate serves as the primary excitatory neurotransmitter. Monitoring spinal glutamate concentrations offers valuable insights into spinal neural processing. Consequently, spinal glutamate concentration has the potential to emerge as a useful biomarker for conditions characterized by increased spinal neural network activity, especially when uptake systems become dysfunctional. In this study, we developed a multichannel custom-made flexible glutamate-sensing probe for the large-animal model that is capable of measuring extracellular glutamate concentrations in real time and in vivo. We assessed the probe's sensitivity and specificity through in vitro and ex vivo experiments. Remarkably, this developed probe demonstrates nearly instantaneous glutamate detection and allows continuous monitoring of glutamate concentrations. Furthermore, we evaluated the mechanical and sensing performance of the probe in vivo, within the pig spinal cord. Moreover, we applied the glutamate-sensing method using the flexible probe in the context of myocardial ischemia-reperfusion (I/R) injury. During I/R injury, cardiac sensory neurons in the dorsal root ganglion transmit excitatory signals to the spinal cord, resulting in sympathetic activation that potentially leads to fatal arrhythmias. We have successfully shown that our developed glutamate-sensing method can detect this spinal network excitation during myocardial ischemia. This study illustrates a novel technique for measuring spinal glutamate at different spinal cord levels as a surrogate for the spinal neural network activity during cardiac interventions that engage the cardio-spinal neural pathway.NEW & NOTEWORTHY In this study, we have developed a new flexible sensing probe to perform an in vivo measurement of spinal glutamate signaling in a large animal model. Our initial investigations involved precise testing of this probe in both in vitro and ex vivo environments. We accurately assessed the sensitivity and specificity of our glutamate-sensing probe and demonstrated its performance. We also evaluated the performance of our developed flexible probe during the insertion and compared it with the stiff probe during animal movement. Subsequently, we used this innovative technique to monitor the spinal glutamate signaling during myocardial ischemia and reperfusion that can cause fatal ventricular arrhythmias. We showed that glutamate concentration increases during the myocardial ischemia, persists during the reperfusion, and is associated with sympathoexcitation and increases in myocardial substrate excitability.

Neuromedin U receptors in GtoPdb v.2023.1

Neuromedin U receptors (provisional nomenclature as recommended by NC-IUPHAR [30]) are activated by the endogenous 25 amino acid peptide neuromedin U (neuromedin U-25, NmU-25), a peptide originally isolated from pig spinal cord [92]. In humans, NmU-25 appears to be the sole product of a precursor gene (NMU, P48645) showing a broad tissue distribution, but which is expressed at highest levels in the upper gastrointestinal tract, CNS, bone marrow and fetal liver. Much shorter versions of NmU are found in some species, but not in human, and are derived at least in some instances from the proteolytic cleavage of the longer NmU. Despite species differences in NmU structure, the C-terminal region (particularly the C-terminal pentapeptide) is highly conserved and contains biological activity. Neuromedin S (neuromedin S-33) has also been identified as an endogenous agonist [97]. NmS-33 is, as its name suggests, a 33 amino-acid product of a precursor protein derived from a single gene and contains an amidated C-terminal heptapeptide identical to NmU. NmS-33 appears to activate NMU receptors with equivalent potency to NmU-25.

The protective effect of total glucosides of white paeony capsules on experimental autoimmune encephalomyelitis

AimsTo learn about the effect and mechanism of total glucosides of white peony capsule (TGP), on experimental autoimmune encephalomyelitis (EAE), an acknowledged animal model of multiple sclerosis (MS). MethodsThe rat model of EAE was induced by subcutaneous injection with guinea pig spinal cord homogenate. The severity of the disease model was assessed by clinical score, hematoxylin and eosin (H&E) and luxol fast blue (LFB). Immunohistochemical assay was used to observe the types of inflammatory cells and adhesive molecule expression. Enzyme-linked immunosorbent assay (ELISA) was applied to detect content of the stem cell growth factor / mast cell growth factor (scf/MGF), interleukin-6 (IL-6) and IL-2. Immunofluorescence assay was applied to observe the expression of connexin43 (Cx43), glial fibrillary acidic protein (GFAP), connexin47 (Cx47) and the monoclonal antibody anti-adenomatous polyposis coli (APC) clone CC1. ResultsCompare with the animals in EAE model group, TGP treated rats (particularly those treated with high doses) showed a significant decrease in morbidity, clinical scores, CNS infiltration of inflammatory cells (including mononuclear macrophages, CD4+ and CD8+ T cells) and demyelination. The key adhesion molecule ICAM-1, cytokines IL-2、IL-6 and scf/MGF were significantly decreased with TGP treatment. Oppositely, PD-1, connexin47 in oligodendrocytes and connexin43 in astrocytes were elevated with TGP treatment. ConclusionTo sum up, TGP exhibited a significantly prevention and treatment effect on EAE rat model, and this improvement was achieved through a combination way composed of glial and inflammatory cells, junction proteins, various factors including adhesion factors, interleukins and scf/MGF.

814 Differences in Human, Pig, and Rat Spinal Cord Stem Cells in Response to Inflammatory and Regenerative Factors In Vitro

INTRODUCTION: The translation of preclinical science to human applications, including regenerative strategies for spinal cord injury, has been poor. Historically, animal models, such as rats and pigs, have been used to model human spinal cord given the difficulty in obtaining live human spinal cord. METHODS: To mimic post-injury inflammation, primary-derived NSPCs from adult humans (n=8), rats (n=6), and pigs (n=5) were treated with interleukin-6 (IL-6), tumor necrosis factor- α (TNFα), or transforming growth factor- β (TGFβ). To direct regeneration, NSPCs were treated with retinoic acid (RA), platelet-derived growth factor (PDGFα), or bone morphogenic protein-4 (BMP4) to induce neurons, oligodendrocytes or astrocytes, respectively. NSPCs were treated for 7 or 14 days and characterized by immunocytochemistry (GFAP, β-iii tubulin, O4). RESULTS: IL-6, TNFα and TGFβ induced astrogenesis of rat NSPCs (3.9±0.7, 5.0±0.9, and 4.0±0.6 fold, respectively) and reduced neurogenesis (0.14±0.90, 0.07±0.04, 0.07±0.05 fold, respectively) after 7 days. Pig NSPCs increased astrogenesis (1.38±0.04, 1.26±0.05, and 1.45±0.04 fold, respectively) after 14 days. Human NSPCs had reduced astrogenesis (0.14±0.07, 0.6±0.2, and 0.12±0.07 fold, respectively) over 14 days, but generated more neurons with IL-6 and TGFβ treatments (1.23±0.05 and 1.34±0.04 fold, respectively). RA increased neurogenesis of human and rat NSPCs, PDGFα increased oligodendrocyte differentiation of rat NSPCs, and BMP4 increased astrogenesis of human and rat NSPCs at low (40ng/mL) and high (100ng/mL) concentrations, respectively. CONCLUSIONS: For the first time, we have compared primary-derived adult spinal cord NSPCs from humans, pigs, and rats and identified differences in their response to pathophysiological and regenerative factors which would impact successful translation of stem cell-based therapies.

Моделювання варіантів транспедикулярної фіксації грудного відділу хребта при резекції одного-трьох хребців

Актуальність. Патологічні переломи хребців є найбільшим ускладненням пухлинного процесу. Застосування додаткової внутрішньої фіксації металевими імплантатами значно покращує результати лікування й скорочує строки реабілітації хворих. Використання транспедикулярних пристроїв забезпечує стабільну фіксацію й стабільність сегментів хребта навіть при максимальному ступені його нестабільності. Мета: визначення оптимального варіанта протяжності заднього спондилодезу при паліативному та радикальному хірургічному лікуванні пухлин хребта. Матеріали та методи. Для вивчення можливостей стабілізації за допомогою транспедикулярної фіксації при резекції одного-трьох хребців провели експериментальні випробування на препаратах хребта свині. Використовували препарати сегментів грудного відділу хребта від Th3 до Th12 хребців. Вивчали 3 групи моделей: з одним резектованим хребцем Th6, з двома — Th5-Th6 і з трьома — Th5-Th7. Результати. Виявлено, що при резекції одного хребця в грудному відділі хребта транс-педикулярна фіксація за схемами 111х111, 101х101 та 011х110 має однакову ефективність. При резекції двох хребців найкращі стабілізуючі можливості мала конструкція за схемою 111хх111, схеми 101хх101 та 011хх110 показали доволі непогану фіксуючу здатність. При резекції трьох хребців грудного відділу хребта найбільш стабільну фіксацію показала схема 111ххх111. Схема фіксації 001ххх100 виявилась нестабільною при всіх видах резекції хребців. Висновки. Експериментальні дослідження транспедикулярної фіксації при радикальному хірургічному лікуванні пухлин хребта, який включає резекцію одного-трьох хребців, показали, що конструкція відповідає вимогам фіксації, коли кріплення відбувається не менше ніж за два верхніх та два нижніх хребці (можна через один), при резекції трьох хребців найбільш стабільною є конструкція з кріпленням за три верхні та три нижні хребці (без пропусків). Система кріплення за один хребець виявилася неспроможною майже при всіх видах резекції.

Exercise training improves memory and produces changes in the adrenal gland morphology in the experimental autoimmune encephalomyelitis.

Objective. The present study sought to verify the effects of an exercise training on the memory along with the morphological assessment of the adrenal gland tissue in the rats with experimental autoimmune encephalomyelitis (EAE). Methods. Female Lewis rats were randomly divided into three groups: EAE group, EAE group with exercise (EAE+Ex), and control group (CO). Each group contained 10 rats. To evaluate the memory, all rats were subjected to the Morris water maze learning test for four consecutive days and one day for a prop test. EAE was induced by guinea pig spinal cord homogenate emulsified in incomplete Freund's adjuvant and heat-mycobacterium. The exercise training on a motorized treadmill was initiated 3 weeks before EAE induction and disconnected 2 weeks post-induction. Results. We found that exercise training for five weeks produced an improved swimming velocity related to memory improvement in EAE+Ex group in comparison with EAE group, but not an incurable adrenal gland tissue after EAE induction. Conclusions. The experimental design selected for this study appears to be an effective treatment for memory in rats with experimental autoimmune encephalomyelitis.

P.235 Differences in Human, Pig, and Rat Spinal Cord Stem Cells in Response to Inflammatory and Regenerative Factors In Vitro

Background: While the use of neural stem/progenitor cells has been reported as a promising therapeutic approach for spinal cord injury, direct comparison of adult primary animal spinal cord NSPCs have not been compared to human NSPCs under the same conditions to characterize intrinsic differences between human/animal NSPC response to inflammatory/regenerative factors. Methods: To mimic post-injury inflammation, primary-derived NSPCs from adult humans, pigs, and rats were treated with pro-inflammatory factors.To direct regeneration, NSPCs were treated with retinoic acid, platelet-derived growth factor or bone morphogenic protein-(BMP4) to induce neurons, oligodendrocytes or astrocytes, respectively.Cultures were treated for 7 or 14 days and characterized by immunocytochemistry. Results: Pro-inflammatory factors promoted more astrogenesis in rat and pig NSPCs compared to human NSPCs and induced neuronal differentiation in human NSPCs. RA increased neurogenesis of human and rat NSPCs, PDGFα increased oligodendrocyte differentiation of rat NSPCs, and BMP4 increased astrogenesis of human and rat NSPCs Conclusions: For the first time, differences in response of human, pig and rat primary NSPCs to inflammatory and regenerative factors have been identified. Better understanding of these differences is essential to improving the successful translation of regenerative therapies to humans.

Architecture and assembly mechanism of native glycine receptors.

Glycine receptors (GlyRs) are pentameric, 'Cys-loop' receptors that form chloride-permeable channels and mediate fast inhibitory signalling throughout the central nervous system1,2. In the spinal cord and brainstem, GlyRs regulate locomotion and cause movement disorders when mutated2,3. However, the stoichiometry of native GlyRs and the mechanism by which they are assembled remain unclear, despite extensive investigation4-8. Here we report cryo-electron microscopy structures of native GlyRs from pig spinal cord and brainstem, revealing structural insights into heteromeric receptors and their predominant subunit stoichiometry of 4α:1β. Within the heteromeric pentamer, the β(+)-α(-) interface adopts a structure that is distinct from the α(+)-α(-) and α(+)-β(-) interfaces. Furthermore, the β-subunit contains a unique phenylalanine residue that resides within the pore and disrupts the canonical picrotoxin site. These results explain why inclusion of the β-subunit breaks receptor symmetry and alters ion channel pharmacology. We also find incomplete receptor complexes and, by elucidating their structures, reveal the architectures of partially assembled α-trimers and α-tetramers.

Application of Autologous Peripheral Blood Mononuclear Cells into the Area of Spinal Cord Injury in a Subacute Period: A Feasibility Study in Pigs.

Simple SummarySpinal cord injury is a medical and social issue causing severe disability. The potential to overcome the consequences of spinal cord injury is related to cell therapy. Peripheral blood is a prospective and available source of cells for further clinical use. In our study, we have evaluated the therapeutic potential of peripheral blood mononuclear cells (PBMCs) on the model of spinal cord injury in pigs. In the subacute period (6 weeks after injury), PBMCs enclosed in fibrin glue were applied into the dorsal area of the injured spinal cord. In this study, we observed that the tissue integrity increased in the area adjacent to the epicenter of injury, and conduction along spinal axons was partially restored after cell therapy in pigs.Peripheral blood presents an available source of cells for both fundamental research and clinical use. In our study, we have evaluated the therapeutic potential of peripheral blood mononuclear cells (PBMCs) excluding the preliminary sorting or mobilization of peripheral blood stem cells. We have evaluated the regenerative potential of PBMCs embedded into a fibrin matrix (FM) in a model of pig spinal cord injury. The distribution of transplanted PBMCs in the injured spinal cord was evaluated; PBMCs were shown to penetrate into the deep layers of the spinal cord and concentrate mainly in the grey matter. The results of the current study revealed an increase in the tissue integrity in the area adjacent to the epicenter of injury and the partially restored conduction along posterior columns of the spinal cord in animals after FM+PBMC application. The multiplex analysis of blood serum and cerebrospinal fluid showed the cytokine imbalance to occur without significantly shifting toward pro-inflammatory or anti-inflammatory cytokine cascades.

Ghrelin Attenuates Neuroinflammation and Demyelination in Experimental Autoimmune Encephalomyelitis Involving NLRP3 Inflammasome Signaling Pathway and Pyroptosis.

Multiple sclerosis (MS) is a chronic autoimmune and degenerative disease of the central nervous system, and conventional treatments have limited efficacy or side effects. Ghrelin, a 28-amino acid octanoylated peptide, has been reported to have neuroprotective effects, including anti-oxidation, anti-inflammation, and anti-apoptosis. Pyroptosis, also called inflammatory cell death, is triggered by overly active inflammasomes and accompanied by the production of numerous cytokines. As immune dysfunction is primarily involved in the pathogenesis of MS, this study aimed to explore the therapeutic effects and precise functional mechanisms of ghrelin against the nod-like receptor protein 3 (NLRP3) inflammasome and pyroptosis in experimental autoimmune encephalomyelitis (EAE). Sprague Dawley rats were immunized with guinea pig spinal cord homogenates and pertussis toxin to develop an EAE model. All rats were randomly divided into four groups: normal control group, EAE group, EAE + ghrelin group, and ghrelin control group. EAE rats showed abnormal behavioral scores and body weight changes. Histologic analysis displayed severe inflammatory infiltration and demyelination in the brain and spinal cord of EAE rats. Ghrelin treatments potently restored these abnormal changes. In addition, the ghrelin-treated EAE group showed significantly downregulated expression of inflammatory cytokines. The expression of proteins involved in the NLRP3 signaling pathway and pyroptosis was decreased as well. We also found that the anti-inflammatory effect of ghrelin was associated with inhibition of nuclear factor (NF)-κB activation. Compared with rats in the healthy control group, rats in the ghrelin control group did not show statistically significant changes in histologic examinations, pro-inflammatory cytokines production, or molecules involved in the NLRP3 signaling pathway, which indicated that ghrelin induced no side effects in the animals of our study. Our findings provide more insight into the use of ghrelin as a novel candidate for MS.

The effects of transplanted mesenchymal stem cells treated with 17-b estradiol on experimental autoimmune encephalomyelitis.

The present study was conducted aimed at exploring the modulatory effects of 17-b estradiol (17-bED) on mesenchymal stem cells (MSCs) in the EAE (experimental autoimmune encephalomyelitis) animal model of multiple sclerosis (MS). Following the isolation of bone marrow-derived MSCs from the bilateral femurs and tibias of the male Wistar rats, the cells were harvested and cultured in the presence of 100nM 17-bED for 24h. EAE was induced in male Wistar rats (8-12weeks old) using guinea pig spinal cord homogenate, in combination with the complete Freund's adjuvant. The MSC therapy was triggered when all of the animals obtained a disability score. The symptoms were monitored on a daily basis throughout the study until the rats were euthanized. The mRNA expression of cytokines, including IL-17, IFN-γ, TNF-α, IL-10, IL-4, and TGF-β together with MMP8 and MMP9 as the family members of matrix metalloproteinases(MMPs) in the brain and spinal cord tissues were examined using real-time PCR. The levels of splenocytes-originated IL-10 and IFN-γ cytokines were also measured by ELISA. The MTT-based research findings showed that the infiltration of lymphocytes into the spleen decreased considerably. It was also observed that the mRNA expression of proinflammatory cytokines decreased significantly, while the mRNA levels of anti-inflammatory cytokines increased remarkably. It was also found that the mRNA levels of the examined matrix metalloproteinases(MMP8 and MMP9) were downregulated significantly. The findings of the present study indicated that the administration of 17-bED enhanced the efficacy of MSCs transplantation and modulated immune responses relatively in the EAE model, via the regulation of either pro- or anti-inflammatory cytokines and matrix metalloproteinases.

Neuromedin U receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Neuromedin U receptors (provisional nomenclature as recommended by NC-IUPHAR [29]) are activated by the endogenous 25 amino acid peptide neuromedin U (neuromedin U-25, NmU-25), a peptide originally isolated from pig spinal cord [90]. In humans, NmU-25 appears to be the sole product of a precursor gene (NMU, P48645) showing a broad tissue distribution, but which is expressed at highest levels in the upper gastrointestinal tract, CNS, bone marrow and fetal liver. Much shorter versions of NmU are found in some species, but not in human, and are derived at least in some instances from the proteolytic cleavage of the longer NmU. Despite species differences in NmU structure, the C-terminal region (particularly the C-terminal pentapeptide) is highly conserved and contains biological activity. Neuromedin S (neuromedin S-33) has also been identified as an endogenous agonist [95]. NmS-33 is, as its name suggests, a 33 amino-acid product of a precursor protein derived from a single gene and contains an amidated C-terminal heptapeptide identical to NmU. NmS-33 appears to activate NMU receptors with equivalent potency to NmU-25.

Comparison of the effects of 17β- estradiol treated and untreated mesenchymal stem cells on ameliorating animal model of multiple sclerosis.

Objectives:The current investigation was undertaken to evaluate the effects of 17β- estradiol (17β-ED) on the potential of the mesenchymal stem cells (MSCs) for modulation of immunity responses in an animal model of multiple sclerosis (MS).Materials and Methods:After isolation of MSCs, cells were cultured in presence of 100 nM 17β-ED for 24 hr. Modeling of experimental autoimmune encephalomyelitis (EAE) was achieved by using guinea pig spinal cord homogenate, in addition to complete Freund’s adjuvant in male Wistar rats. The processes of cell therapy were started following 12 days post-immunization. This duration allows all animals to develop a disability score. The achieved EAE clinical symptoms were regularly monitored every day until day 36, when all of examined rats were euthanized. Results:Cell therapy in the EAE rats with 17β-ED-primed MSCs exhibited more desirable consequences, which in turn lead to regression of the cumulative clinical score and neuropathological changes that are more than the therapy with untreated MSCs. The serum measures of myeloperoxidase (MPO), nitric oxide (NO) as well as splenocytes-originated pro-inflammatory interleukin-17 (IL-17) and tumor necrosis factor alpha (TNF-α) were significantly decreased in EAE rats treated by 17β-ED primed-MSCs compared to EAE rats that received untreated MScs. Conclusion:Combination of 17β-ED and MSCs more effectively improved the signs and symptoms of EAE.

Toward the monitoring of the spinal cord: A feasibility study

The usual imaging techniques providing only limited information, there is an unmet need for methods, firstly, to monitor in real time the functional status of the Spinal Cord (SC) and, secondly, to assess the functional consequences of the SC Injuries (SCI) and the effect of therapeutic interventions. To meet these needs, our project aims to investigate a new imaging approach, through the realization of a device for measuring the SC activity. This approach will establish locally and specifically the functional state of the SC in real time, which will provide a breakthrough in surgery, as well as in the care and therapeutic trials for SCI.This paper presents the first results of this research project on pig subjects, focusing on the opto-electrical part, based on Near-InfraRed Spectroscopy (NIRS). Firstly, the pig SC optical characterization was performed in different conditions, using a portable spectrophotometer in order to define the wavelengths of interest and their corresponding optical attenuation. In transmission mode, the Optical Density (OD) was evaluated between 3.5 and 6.5 in the [500; 950] nm range. In reflection mode, a figure between 11% and 33% is obtained in the same wavelength range. We find that wavelengths between 600 nm and 940 nm are good candidates to monitor the SC functional activity. Secondly, thanks to these results, a specific opto-electrical system has been designed for the transmission mode only, with adapted light sources and custom probes with its front-end to observe the autonomic functions in the SC. Results on the measured haemodynamic variations, at rest and under stimulation, show in real time the impact of a global stimulus on a local section of the SC. However, with a low AC-to-DC ratio (around 1%), the SC Photo PlethysmoGram (PPG) acquisition isn't simple and the best trade-off between power consumption and Signal-to-Noise Ratio (SNR) must be found in the perspective of the Embedded System (ES) development.This study demonstrates, for the first time, the feasibility of the SC activity monitoring using NIRS in transmission mode in a big animal model, where the perfect alignment of the light emitter and receptor isn't necessary due to the diffusive property of the biological media. It contributes to heading towards the use of the Internet of Things (IoT) for medical applications, through the monitoring of the SC during highly invasive processes, such as the stabilization of the spine, in the form of implants and other surgeries, such as that of the aorta, with the use of specific minimally invasive catheters.

Nicotine Augments the Beneficial Effects of Mesenchymal Stem Cell-based Therapy in Rat Model of Multiple Sclerosis

ABSTRACTExperimental autoimmune encephalomyelitis (EAE) in rats through immunization with guinea pig spinal cord homogenate (GPSCH) produces a chronic disease with a relapsing pattern such as multiple sclerosis (MS) in humans. In previous studies, the immunomodulatory benefits of mesenchymal stem cells (MSCs) and nicotine have already been determined. Thus, this research was conducted to assess the additional benefits of the combination therapy of MSCs and nicotine in a rat model of MS. EAE was induced by GPSCH and complete Freund’s adjuvant (CFA) in female Wistar rats. The therapies were initiated at day 12 post-immunization (p.i.), when the rats developed a neurological disability score. The symptoms were recorded daily until day 33, when the rats were sacrificed. Finally, the splenocytes were evaluated by Enzyme-linked immunosorbent assay (ELISA) for cytokine production. The therapeutic treatment in the EAE rats with a combination of MSCs and nicotine exhibited a more desirable outcome, causing the regression of the average mean clinical score and neuropathological features to be more favorable than the treatment with either therapy alone. The combination therapy led to a significant reduction in the cumulative disease disability from day 21. For the EAE rats treated with nicotine and MSCs, this period was started from day 22 and 28 p.i., respectively. Besides the increase in the levels of IL-10, the combined therapy significantly reduced the splenocytes production of pro-inflammatory IL-17 as well as TNF-α more profoundly than either of the medications alone. In conclusion, the combination of MSCs and nicotine can be suggested as a promising strategy for further MS therapeutics improvement.

Determination of stable reference genes for RT-qPCR expression data in mechanistic pain studies on pig dorsal root ganglia and spinal cord

RNA expression levels for genes of interest must be normalised with appropriate reference or “housekeeping” genes that are stably expressed across samples and treatments. This study determined the most stable reference genes from a panel of 6 porcine candidate genes: beta actin (ACTB), beta-2-microglobulin (B2M), eukaryotic elongation factor 1 gamma-like protein (eEF-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), Ubiquitin C (UBC) in sacral dorsal root ganglia and spinal cord samples collected from 16 tail docked pigs (2/3rds of tail amputated) 1, 4, 8 and 16weeks after tail injury (4 pigs/time point). Total RNA from pooled samples was measured by SYBRgreen real-time quantitative PCR. Cycle threshold values were analysed using geNorm, BestKeeper and NormFinder PCR analysis software. Average expression stability and pairwise variation values were calculated for each candidate reference gene. GeNorm analysis identified the most stable genes for normalisation of gene expression data to be GAPDH>eEF-1>UBC>B2M>ACTB>SDHA for dorsal root ganglia and ACTB>SDHA>UBC>B2M>GAPDH>eEF-1 for spinal cord samples. Expression stability estimates were verified by BestKeeper and NormFinder analysis. Expression stability varied between genes within and between tissues. Validation of most stably expressed reference genes was performed by normalisation of calcitonin gene related polypeptide beta (CALCB). The results show similar patterns of CALCB expression when the best reference genes selected by all three programs were used. GAPDH, eEF-1 and UBC are suitable reference genes for porcine dorsal root ganglia samples, whereas ACTB, SDHA and UBC are more appropriate for spinal cord samples.

Diffusion time dependence of microstructural parameters in fixed spinal cord

Biophysical modelling of diffusion MRI is necessary to provide specific microstructural tissue properties. However, estimating model parameters from data with limited diffusion gradient strength, such as clinical scanners, has proven unreliable due to a shallow optimization landscape. On the other hand, estimation of diffusion kurtosis (DKI) parameters is more robust, and its parameters may be connected to microstructural parameters, given an appropriate biophysical model. However, it was previously shown that this procedure still does not provide sufficient information to uniquely determine all model parameters. In particular, a parameter degeneracy related to the relative magnitude of intra-axonal and extra-axonal diffusivities remains. Here we develop a model of diffusion in white matter including axonal dispersion and demonstrate stable estimation of all model parameters from DKI in fixed pig spinal cord. By employing the recently developed fast axisymmetric DKI, we use stimulated echo acquisition mode to collect data over a two orders of magnitude diffusion time range with very narrow diffusion gradient pulses, enabling finely resolved measurements of diffusion time dependence of both net diffusion and kurtosis metrics, as well as model intra- and extra-axonal diffusivities, and axonal dispersion. Our results demonstrate substantial time dependence of all parameters except volume fractions, and the additional time dimension provides support for intra-axonal diffusivity to be larger than extra-axonal diffusivity in spinal cord white matter, although not unambiguously. We compare our findings for the time-dependent compartmental diffusivities to predictions from effective medium theory with reasonable agreement.

Neuroimmunomodulation by allogeneic seminal vesicle fluid in CNS is sex-independent

Objectives: Thymus-arisen FoxP3 regulatory T cells (Tregs) are one of the most important immunoregulatory mechanisms in the central nervous system (CNS) and pregnancy. Multiple sclerosis (MS) is an inflammatory disease of CNS associated with a reduced frequency and/or function of Tregs. Previous works have shown that seminal vesicle fluid affects female immune system and causes expansion of Tregs pool in the female reproductive tissue upon mating. Accordingly, it has been demonstrated that intra-CSF administration of seminal vesicle fluid from Wistar rats can ameliorate clinical sign of a female Lewis rat model of experimental autoimmune encephalomyelitis (EAE), the animal model of MS. The results indicated an up-regulation of FoxP3 expression in the brain and spinal cord. However, there are sex-based differences in the CNS structure & composition, sex hormones influence immune system, and gender-based differences affect treatment response. Therefore, we decided to find out if anti-inflammatory effect of seminal vesicle fluid is sex-dependent or -independent.Methods: EAE was induced in male Lewis rats using guinea pig spinal cord and complete Freund’s adjuvant. Intra-CSF injection was done on day 7 after EAE induction and the animals were followed-up until on day 14 after EAE induction when sacrificed. Then, brain and spinal cord of the animals were isolated, total RNA was extracted, and expression of mRNA for IFN-γ, IL-4, IL-17, and FoxP3 was determined using real-time PCR where β-actin was used as reference gene.Result: demonstrated that intra-CSF administration of seminal vesicle fluid from male Wistar rats ameliorated EAE in male Lewis rats and increased FoxP3 in the brain and spinal cord.Conclusion: This study suggests that seminal vesicle fluid from Wistar rats has anti-inflammatory effect on Lewis rats in a sex-independent manner. In addition, seminal vesicle fluid from Lewis rats had not beneficial effect on EAE in male Lewis rats. This is consistent with Tregs increase in allogeneic mating. More research is required to find out the immunologic aspect of allogeneic versus syngeneic administration of seminal vesicle fluid.

Pertussis toxin promotes relapsing–remitting experimental autoimmune encephalomyelitis in Lewis rats

Animal models simulate different aspects of human diseases and are essential to get a better understanding of the disease, studying treatments and producing new drugs. Experimental autoimmune encephalomyelitis (EAE) is a preferred model in multiple sclerosis research. Common EAE model in Lewis rats is induced using MBP peptide as a myelin antigen which results in a monophasic disease course. In the present study, EAE was induced in Lewis rats by homogenized guinea pig spinal cord along with or without pertussis toxin (PT). When PT was used, EAE turned into remitting–relapsing form and worsen the clinical symptoms. Higher inflammation and oxidative stress marker gene expression was observed when PT was administrated.