Rat Spinal Tissue Research Articles

LncRNA SNHG1 attenuates neuropathic pain following spinal cord injury by regulating CDK4 level.

Neuropathic pain (NP) is one of the most intractable complications of spinal cord injury (SCI). This study aims to explore the role of long non-coding RNA (lncRNA) SNHG1 in influencing SCI-induced NP. After establishment of the spinal nerve ligation (SNL) model in rats, spinal tissues were extracted. SNHG1 level in rat spinal tissues was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The role of SNHG1 in the development of NP was explored by assessing paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in model rats. The interaction between SNHG1 and CDK4 was explored by Luciferase assay and RIP (RNA-Binding Protein Immunoprecipitation). Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR were conducted to determine inflammatory factor levels in rat spinal tissues. SNHG1 was upregulated in rats undergoing SNL. Knockdown of SNHG1 alleviated the development of NP and overexpression of SNHG1 was capable of inducing NP symptoms in uninjured rats. SNHG1 induced NP by directly regulating CDK4 level. SNHG1 is a novel target in the treatment of NP associated with neuroinflammation.

Immunolocalization of Keratan Sulfate in Rat Spinal Tissues Using the Keratanase Generated BKS-1(+) Neoepitope: Correlation of Expression Patterns with the Class II SLRPs, Lumican and Keratocan.

This study has identified keratan sulfate in fetal and adult rat spinal cord and vertebral connective tissues using the antibody BKS-1(+) which recognizes a reducing terminal N-acetyl glucosamine-6-sulfate neo-epitope exposed by keratanase-I digestion. Labeling patterns were correlated with those of lumican and keratocan using core protein antibodies to these small leucine rich proteoglycan species. BKS-1(+) was not immunolocalized in fetal spinal cord but was apparent in adult cord and was also prominently immunolocalized to the nucleus pulposus and inner annulus fibrosus of the intervertebral disc. Interestingly, BKS-1(+) was also strongly associated with vertebral body ossification centers of the fetal spine. Immunolocalization of lumican and keratocan was faint within the vertebral body rudiments of the fetus and did not correlate with the BKS-1(+) localization indicating that this reactivity was due to another KS-proteoglycan, possibly osteoadherin (osteomodulin) which has known roles in endochondral ossification. Western blotting of adult rat spinal cord and intervertebral discs to identify proteoglycan core protein species decorated with the BKS-1(+) motif confirmed the identity of 37 and 51 kDa BKS-1(+) positive core protein species. Lumican and keratocan contain low sulfation KS-I glycoforms which have neuroregulatory and matrix organizational properties through their growth factor and morphogen interactive profiles and ability to influence neural cell migration. Furthermore, KS has interactive capability with a diverse range of neuroregulatory proteins that promote neural proliferation and direct neural pathway development, illustrating key roles for keratocan and lumican in spinal cord development.

Differential densimetry: A method for determining ultra-low fluid flux and tissue permeability

Osmotic transport devices (OTDs) are forward osmosis membrane devices that we recently developed to remove accumulated fluid from swollen tissue, in-vivo, under severe conditions. As such, the relative volume of the fluid required to be removed and the volumetric flowrate may be two orders of magnitude less than the operating volume and tangential flowrate of the device. This makes it challenging to measure the rate of fluid flow from the swollen tissue. Here, we introduce a differential densimetry method for determining ultra-low volumetric flux through tissue samples. This technique uses two vibrating tube density sensors, one placed upstream of the membrane in contact with the tissue sample, and one placed downstream. Any flow of biological fluid withdrawn through the tissue will combine with the OTD operating fluid resulting in an observed density shift in the second density sensor. By measuring the difference in density between the upstream and downstream fluids, one can calculate the amount of fluid flowing across the tissue with a relatively high level of sensitivity. This method is also relatively insensitive to drift from temperature fluctuations and capable of continuously monitoring tissue permeability in real time. As a proof of concept, we used this technique to measure fluid flow across ex-vivo rat spinal tissue for an appropriately scaled OTD. The repeatability error had a maximum of only 12%. This implies that this method can provide highly acceptable flux measurements with reasonable reproducibility in real-time applications of fluid removal in-vivo.

SpinoMimetic CryoSponges as advanced biomaterial archetypes for enhanced neuronal anchoring and support: Achievement of maximal mechanotransduction via minimal functionalization and crosslinking

Event Abstract Back to Event SpinoMimetic CryoSponges as advanced biomaterial archetypes for enhanced neuronal anchoring and support: Achievement of maximal mechanotransduction via minimal functionalization and crosslinking Pradeep Kumar1, Yahya E. Choonara1, Girish Modi2 and Viness Pillay1 1 University of the Witwatersrand, Wits Advanced Drug Delivery Platform Research Unit, South Africa 2 University of the Witwatersrand, Department of Neurology, South Africa Introduction: Mechanical properties of a biomaterial scaffold play a definitive role in neuronal support, differentiation and even regeneration [1]. To achieve neuromimetism, mechanotransduction through focused selection of biomaterials (including blends) require fine-tuning of their chemical morphology [2]. In this study, novel “SpinoMimetic CryoSponge Architectures” were developed employing various polyethylene glycol (PEG) derivatives (2kDa) with chitosan network for potential and enhanced axonal anchoring. Methods: Genipin-crosslinked chitosan/PEG cryosponges were fabricated via focused graded functionalization of methoxy-PEG (mPEG) and blending with chitosan to obtain chitosan-blend-mPEG (CbPEG-OH), chitosan-blend-mPEG-CHO (CbPEG-CHO), chitosan-blend-mPEG-NH2 (CbPEG-NH2) and chitosan-blend-mPEG-COOH (CbPEG-COOH). The scaffolds were developed via a unique concurrent blending-crosslinking-cryogelling technique followed by incubation at -20°C and finally thawing at room temperature. Results and discussion: Morphologically, 7.5mm diameter cylindrical CbPEG-OH, CbPEG-CHO, CbPEG-NH2 and CbPEG-COOH cryosponges composed of macroporous (discontinuous), uniaxial macroporous (continuous), mesoporous (continuous) and microporous (continuous) channels, respectively, of varying surface areas. The matrix resilience of the hydrated samples (at 25% strain) ranged between 12.7-17.5% on a force-time scale. The mechanical properties of CbPEGs prepared were within the range of biomechanical matrix rigidity of the rat spinal tissue tested (nanotensile testing). The neuronal anchoring of PC12 cells on the scaffolds was deduced via a relationship between the inherent matrix resilience (Rm; %), surface area (Ap) and solution viscosity (η) of the SpinoMimetic CryoSponges. At constant base/conjugate polymer ratio: Ap ∝ √ η/Rm (Equation 1) Ap = ϸ √ η/Rm (Equation 2) where, þ (m4s4/kg) is Mechano-Mesoporous Flow Coefficient (novel biomaterial coefficient) which is a function of end group functionality of the conjugate polymer and expresses the proliferation and diffusion behaviour of PC12 cells dependent on local cytomechanical signals. The mathematical propositions in terms of þ explained the spatiotemporal evolution of the growth factor concentration and neuronal connectivity (CbPEG-CHO > CbPEG-OH > CbPEG-NH2 > CbPEG-COOH). The neuronal anchoring performance of various cryosponges along with the energy relationships employing static lattice atomistic simulations (AMBER force field) demonstrated a significant role of torsional contributions arising from deviations from optimum dihedral angles as a function of degree of crosslinking. Conclusion: A novel relationship is hereby introduced for the prediction of behavior of axonal regeneration in response to substrate resilience, mechanical stress and topographic features of scaffolds with far reaching implications in spinal cord injury intervention. National Research Foundation (NRF) of South Africa

Effect of nerve growth factor on three-dimensional culture of adult rat spinal tissue embedded in the hydrogel PuraMatrix

Effect of nerve growth factor on three-dimensional culture of adult rat spinal tissue embedded in the hydrogel PuraMatrix

The time course of hydroxyl radical formation following spinal cord injury: the possible role of the iron-catalyzed Haber-Weiss reaction.

This study explores whether the hydroxyl radical (*OH)-one of the most destructive reactive oxygen species-plays a role in secondary spinal cord injury (SCI). First, we measured the time course of *OH formation in rat spinal tissue after impact SCI by administering salicylate as a trapping agent into the intrathecal space of the cord and measuring the hydroxylation products of salicylate, 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-DHBA) by HPLC. The 2,3-DHBA concentration was significantly higher in injured spinal tissue than in sham controls at 5 min, 1 and 3 h, but not at 5 h post-injury. Second, we generated *OH by administering H(2)O(2) and FeCl(2)/EDTA (Fenton's reagents) at the concentrations produced by SCI into the gray matter of the cord for 4 h and found that it induced significant cell loss at 24 h post-*OH exposure. Mn (III) tetrakis (4-benzoic acid) porphyrin(MnTBAP)-a broad spectrum reactive species scavenger-significantly reduced *OH-induced cell death. Finally, we generated superoxide and administered FeCl(3)/EDTA in the intrathecal space of the cord at the concentration produced by SCI and measured extracellular *OH formation in the gray matter of the cord by microdialysis sampling. We found that the levels of *OH significantly increased compared to the pre-administration level, indicating that *OH can be produced in vivo by the iron-catalyzed Haber-Weiss reaction. All together, we demonstrated that *OH is an endogenous secondary damaging agent following SCI and the metal-catalyzed Haber-Weiss reaction may contribute to early *OH formation after SCI.

Pregabalin and gabapentin reduce release of substance P and CGRP from rat spinal tissues only after inflammation or activation of protein kinase C

Gabapentin and pregabalin are amino acid derivatives of γ-amino butyric acid that have anticonvulsant, analgesic, and anxiolytic-like properties in animal models. The mechanisms of these effects, however, are not well understood. To ascertain whether these drugs have effects on sensory neurons, we studied their actions on capsaicin-evoked release of the sensory neuropeptides, substance P and calcitonin gene-related peptide from rat spinal cord slices in vitro. Although release of immunoreactive peptides from non-inflamed animals was not altered by either drug, prior in vivo treatment by intraplantar injection of complete Freund's adjuvant enhanced release from spinal tissues in vitro, which was attenuated by gabapentin and pregabalin. These drugs also reduced release of immunoreactive neuropeptides in spinal tissues pretreated in vitro with the protein kinase C activator, phorbol 12,13-dibutyrate. Our results suggest that gabapentin and pregabalin modulate the release of sensory neuropeptides, but only under conditions corresponding to significant inflammation-induced sensitization of the spinal cord.