Mechanobiology Article Review and Analysis

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Review and analysis of the biomedical and kinematic properites of tensile strength within the spine during elongation

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Mechanobiology Article Review and Analysis

  1. 1. Probing the Influence of Myelin and Glia on the Tensile Properties of the Spinal Cord DeAndria Hardy December 2, 2008
  2. 2. Traditional medical data, until this point, concerning the glia matrix has primarily focused on classifying it as merely a structural component. The article reviewed sought to expand on traditional knowledge. Researchers David Shreiber, Hailing Hao, and Ragi Elias proposed that the glia matrix in connection with the myelin sheath contributes to the overall mechanical properties present within the spinal cord. Shreiber et al hypothesized that a disrupted glia matrix and a demyelinated spinal cord decreased the spinal cord’s stiffness and ultimate tensile stress. To evaluate this conjecture the researchers performed an experiment involving uniaxial tensile testing of chick embryos on day 18 of development. The experiment was borne out of a desire of the researchers to expand on the accepted classification of glia. The first step in this process was to compile characteristics of glia from known data and establish the proper niche for the results of the experiment’s hypothesis. The glia matrix is composed of non-neuronal cells. These cells along with being the binding force among neurons provide many regulatory functions for the central nervous system. The glia matrix provides nutrition, it maintains homeostasis, and forms myelin. These cells are broken down into divisions of the central nervous system
  3. 3. (CNS): astrocytes, oligodendrocytes, radial glia, and ependymal cells. The researchers focused exclusively on astrocytes and oligodendrocytes. These particular cells contribute the most to structural “cellular scaffolds” of the glia matrix (Schultze 1866). Mechanical properties of the spinal cord are greatly impacted by the scaffolds. They increase the tissues of the CNS’s ability to withstand loading and inherent stresses. In general load bearing tissues do not include those of the CNS. These tissues are commonly accepted as bones, tendons, and blood vessels. That is because these tissues experience continual loading during everyday activities and they accept and disperse the load to reduce damage to the human body. Observing this as the case for load bearing tissues, Shreiber et al. relied on the previous research of Qing Yuan to draw the following: glia could be considered as a load bearing tissue because during everyday activities where the flexion occurs in the spinal cord it endures a 6-10% strain and certain glia cells protect against this flexion to reduce pressures and abnormal forces from causing the spinal cord injury. The particular glia cells responsible for the dispersion of stresses are the oligodendrocytes and astrocytes. These cells accomplish this by creating a “cellular crosslink”. The crosslink stems from the
  4. 4. interconnections present between oligodendrocytes and axons and astrocytes and blood vessels. Once it was established that the mechanical properties of the spinal cord were a result of the “cellular crosslink” the researchers had to choose the proper method to disrupt this network. The experiment’s hypothesis required a method that would target the three components in question: astrocytes, oligodendrocytes, and myelin. The researchers were forced to rely on two methods to accomplish the desired disruptions (Graca and Blakemore 1986). The first method involved a chemical interference using ethidium bromide (EB), an agent that is cytotoxic to oligodendrocytes and astrocytes. Ethidium bromide selectively targets dividing cells and leaves cells like neurons intact. The other method was immunological. This method used galactocerebroside (GalC), an antibody that specifically targeted myelin producing oligodendrocytes but did not cause astrocyte damage. The first step of the experiment was to introduce the agents for myelin suppression. There were four reagents involved, two for suppression and two as controls. To inject the reagent into the chick eggs, small windows were created in the shells. After the injections the windows were sealed with cellophane tape until stage E18 of
  5. 5. development. Chick eggs at stage E14 were injected with either 0.01% EB in 0.1% saline or its control 0.1% saline. An Immunoglobulin G (IgG) rabbit-αGalC antibody at a 1:25 dilution with a 20% serum complement in 0.1M Phosphate Buffered Saline (PBS) was injected into chick eggs at stage E12. Its control was a pure rabbit IgG at a 1:25 dilution with a serum complement solution. Each chick egg received two 3µl injections, one in the cervical spinal cord and one into the thoracic spinal cord. At stage E18 the spinal cords from each of the chick eggs were extracted for testing. Upon excising the spinal cords at stage E18, they were quantified for myelination. The researchers used myelin basic protein (MBP) immunohistochemistry and osmium tetroxide treatment for the quantifications. Three spinal cords from each of the four experimental conditions were examined. From each spinal cord five sections were taken and five areas within the white matter were selected at random for testing. The immunohistochemistry quantifications were performed by first harvesting the stage E18 spinal cords and immersion fixed in 4% paraformaldehyde. The spinal cords were then incubated in a 20% sucrose-saline solution at 4oC overnight. The next day longitudinal sections were cut from the frozen spinal cords
  6. 6. with a cryostat. The sections were labeled with a 1:400 dilution of rabbit anti-MBP and a 1:100 dilution of mouse α-NeuroFilament-200 (Sigma). Then the sections were incubated in a goat anti-rabbit Alexa 488 dye and goat anti-mouse Alexa 546 dye. The Alexa 488 was used to visualize the MBP and the Alexa 546 was used to visualize the neurofilaments. The osmium tetroxide quantifications were performed by placing 5µm frozen transverse sections on microslides that were pre-treated with a 2% osmium tetroxide solution for 30 minutes and then dehydrated by an alcohol wash. The slides were coverslipped and the myelin sheaths were counted at high magnification under a brighfield microscope. The number of myelin sheaths were averaged for each slide, spinal cord, and experiment condition. The data was then normalized to the control condition. To test the role of glia in the mechanical properties of the spinal cord, each of the chick spinal cords were stretched uniaxially at a low strain rate until failure. The excised stage E18 spinal cords were exposed ventrally and an 11 segment section that extended from the first nerve root was measured. The measurement was performed three times for accuracy. The dorsal half of the vertebrae was then removed and the spinal cord re-measured. This too
  7. 7. was performed three times. The resulting section was visually checked for any damages that could have occurred during excising. The spinal cords were then marked off by reflective plastic dots into a 12mm section. Three additional dots were added as a means of monitoring uniformity during testing. Afterward the spinal cords were placed in a Bose/Enduratec ELF 3200 with a 0.5N cantilever load cell for uniaxial testing. The ends of the spinal cords were placed on polyethylene plates that were 10mm apart of the load cell crossheads, with the plastic dots marking the 12mm section exactly at the edge as seen in Figure 1. Each spinal cord was stretched once at 0.012mm/s with a .001 s-1 strain rate. Images were taken every .5mm to assess the uniformity of the strain. The load and displacement of each spinal cord were recorded at 1.67 Hz and then converted to a nominal stress. The stress-stretch curves were plotted and the ultimate tensile stress, σUTS, and the stretch at the ultimate tensile stress, λUTS, were identified. The results showed that after injection the spinal cords treated with EB and αGalC were significantly shorter than the controls (Table 1). In general the spinal cords injected with the IgG or saline controls exhibited similar
  8. 8. MBP immunoreactivity to embryos without any treatment. While the other spinal cords injected with EB and αGalC exhibited a decrease in immunoreactivity or a decrease in the number of detectable myelinated axons (Figure 2). Immunohistochemistry was use to asses the demyelinated axons. Alpha-Glial Fibrillary Acidic Protein (α-GFAP) was used to stain and test for astrocytes. GalC was used to stain and test for oligodendrocytes. The green immunofluorescence in Figure 2e, 2b, and 2d shows the experiment control. In Figure 2a and 2c there is a red immunofluorescence. This exhibits the myelin decrease. The results of the uniaxial testing illustrated nonlinear, strain stiffening behavior (Graph 1). This behavior was made apparent when each condition was fit to the Ogden strain energy potential function: W = 2G/α2 (λα1+ λα2 + λα3− 3). The intermittent peaking on the graphs depicts microfractures and recovery within the spinal cords. Even after the ultimate tensile stretch is achieved these intermittent peaks can be seen, showing that the spinal cord was still attempting to accept and redistribute the loading on it. The graph also depicts significantly lower ultimate stress for the spinal cords treated with EB and
  9. 9. αGalC. The two treatment conditions of EB and αGalC express a significantly lower shear modulus (Table 2). All of the results from the uniaxial tensile testing and subsequent calculations verified the researchers’s hypothesis that glia is more than just a binding element of the CNS. The assumption that if the glia matrix indeed contributed to the mechanical properties of the spinal cord a disruption would decrease the overall ultimate tensile stress of the spinal cord was quantified. In experimental conditions where the primary components of the glia matrix, astrocytes and oligodendrocytes, were interrupted a substantial decrease presented itself. When critiquing this experiment that researcher’s success is an obvious positive note. More impressive than the experiment’s success is the niche in which the researcher’s chose. Amidst all of the current research that exist concerning demyelination and the spinal, as it relates to diseases like Multiple Sclerosis, the researchers explored it mechanical properties. It is commendable to go against the norm of signal transduction and myelin. The researchers also chose a different form of deformation than any previous experiment. Other experiments done on the spinal cord have been in reference to force present on the spinal cord with compressive or shear
  10. 10. forces. Shreiber et al. chose to test in tension versus the other accepted methods. This gave the experiment and its result validity separate from conclusion drawn from other experiments. The experiment’s methodology was also impressive. The experiment did not rely on just one method to demyelinate the spinal cord. By using both a chemical and immunological method of disruption, it eliminated skewed results. For instance, if the researchers only used one method of disruption the results could be called into question because the results were all inclusive. Also the method was very precise and easily reproduced. In review of other experiments, the methods are so tedious and materials are so difficult to acquire it become almost impossible to recreate the experiment. Finally, the most notable aspect of the experiment is that is has the ability for other avenues of research. This research provides a “leaping point” for other research to explore remyelination. Current research has focused on remyelination solely to assist in signal transduction. This research opens the opportunity to explore how remyelination affects the stability and other mechanical properties of the spinal cord. It could lead to other discoveries on how to improve the quality of life for
  11. 11. those who suffer from demyelinating diseases like Multiple Sclerosis. Figure 1- Schematic of uniaxial testing setup Figure 2- Immunoreactivity of experiment conditions
  12. 12. Table 1- Length and area measurements of stage E18 spinal cords EB (n=6) Length (mm) Area (mm2) Saline (n=5) αGalC (n=5) IgG (n=5) Control (n=6) 22.35±0.2 22.83±0.25 21.99±0.35 22.89±0.36 23.03±0.49 1.47±0.05 1.54±0.04 1.28±0.04 1.58±0.11 1.51±0.02 Graph 1- Stress-stretch curves for various experiment conditions Table 2- Results of uniaxial testing σUTS λUTS G(kPa) α EB 28.4 ± 9.38 1.38 ± 0.09 17.4 ± 5.70 8.32 ± 2.55 Saline 77.8 ± 19.6 1.43 ± 0.09 29.2 ± 7.38 8.49 ± 1.34 αGalC 55.9 ± 28.9 1.45 ± 0.10 17.7 ± 6.80 8.74 ± 0.84 IgG 93.5± 37.3 1.45 ± 0.10 30.0 ± 7.26 9.00 ± 1.62 Graph 2- Stress-stretch comparison curves Control 85.2 ± 17.7 1.42 ± 0.03 32.8 ± 9.53 8.22 ± 1.27
  13. 13. References Bain AC, Shreiber DI, Meaney DF (2003) Modeling of microstructural kinematics during simple elongation of central nervous system tissue. J Biomechanical Engineering 125(6): 798–804. Elias Ragi, Hao Hailing, David Shreiber (2008) Probing the influence of myelin and glia on the tensile properties of the spinal cord. Biomedical Model Mechanobiology Graca DL, Blakemore WF (1986) Delayed remyelination in rat spinal cord following ethidium bromide injection. Neuropathology Applied Neurobiology 12(6):593–605. Schultze M (1866).Zur Anatomie und Physiologe der Retina. Max Cohen & Sons, Bonn

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