Exceptional mechanical and electrical properties of carbon nanotubes (CNT) have attracted neuroscientists and neural tissue engineers aiming to develop novel devices that interface with nervous tissues. In the central nervous system (CNS), the perinatal chloride shift represents a dynamic change that forms the basis for physiological actions of γ-aminobutyric acid (GABA) as inhibitory neurotransmitter, a process of fundamental relevance for normal functioning of the CNS. Low intra-neuronal chloride concentrations are maintained by chloride-extruding transporter, potassium chloride cotransporter 2 (KCC2). KCC2's increasing developmental expression underlies the chloride shift. In neural injury, repressed KCC2 expression plays a co-contributory role by corrupting inhibitory neurotransmission. Mechanisms of Kcc2 up-regulation are thus pertinent because of their medical relevance, yet they remain elusive.
In view of known nanoscopic size, extraordinary strength and electrical conductivity of carbon nanotubes (CNT), (Lee, W., Parpura, V. (2009) Prog. Brain Res. 180:110-125; Lu, J. et al. (2008) Nano Lett. 8:3325-3329; Odom, T. W. et al. (2002) Ann. NY Acad. Sci. 960:203-215) whether CNT exposure alters neurons' gene expression so that their cell- and network physiological properties could be modified was investigated. One suitable and highly informative neuronal cell-physiological parameter is intraneuronal chloride, which dictates neurons' transmission in response to GABA and glycine, known to be inhibitory in mature brain and spinal cord, and excitatory in neural development. (Boulenguez, P. et al. Nat Med. 16:302-307; Coull, J. A. et al. (2003) Nature 424:938-942; Delpire, E., Mount, D. B., (2002) Annu Rev Physiol, 64:803-843; Fiumelli, H., Cancedda, L., Poo, M. M. (2005) Neuron 48:773-786; Ganguly, K. et al. (2001) Cell 105:521-532; Hubner, C. A. et al. (2001) Neuron 30:515-524; Malek, S. A., Coderre, E., Stys, P. K. (2003) J. Neurosci 23:3826-3836; C. et al. (2011) J. Physiol 589:2475-2496; Price, T. J., Cervero, F., de Koninck, Y. (2005) Top Med Chem 5: 547-555; Rivera, C. et al. (1999) Nature 397:251-255; Yeo, M. et al. (2009) Neurosci 29:14652-14662; Woo, N. S. et al. (2002) Hippocampus 12:258-268; Zhu, L. et al. (2008) Epilepsy Res 79:201-212). The perinatal chloride shift is a profound developmental transformation of CNS neurons as they change from migratory phenotype to synaptic connectivity. Bortone, D., Polleux, F. (2009) Neuron 62: 53-71. Developmentally earlier intraneuronal chloride renders GABA excitatory, which is converted into the mature of low intraneuronal chloride by transcriptional upregulation of the chloride-extruding transporter KCC2. (Ganguly, K. et al. (2001) Cell 105:521-532; Yeo, M. et al. (2009) J. Neurosci 29:14652-14662). Low intraneuronal chloride sustains inhibitory GABA-ergic neurotransmission in neural circuits, a quintessential function of the vertebrate CNS. Aside from its basic relevance, a reversal of the developmental chloride shift has been demonstrated the pathogenesis of diseases with a common feature of neural injury, namely epilepsy, pain, traumatic brain injury and cerebral ischemia. (Pellegrino, C. et al. (2011) J. Physiol 589:2475-2496; Price, T. J., Cervero, F., de Koninck, Y. (2005) Curr Top Med Chem 5: 547-555).
The present disclosure is based in part on the novel finding that that primary cortical neurons, cultured on high-conductivity few-walled CNT (Qi, H., Qian, C., Liu, J. (2006) Chemistry of Materials 18: 5691-5695) have a strikingly accelerated chloride shift caused by increased KCC2 expression, where the KCC2 upregulation was fully dependent on neuronal voltage-gated calcium channels.
One aspect of the present disclosure provides a substrate comprising, consisting of, or consisting essentially of high-conductivity few-walled CNTs dispersed thereon, wherein the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm. In certain embodiments, the CNTs comprise an intrinsic electrical conductivity in a range of about 1,000 S/cm to about 3,000 S/cm. In an alternative embodiment, the CNTs compromise an intrinsic electrical conductivity in a range of about 1,500 S/cm to about 2,500 S/cm.
Another aspect of the present disclosure provides a method of upregulating KCC2 expression in a neuron comprising culturing the neuron on substrate comprising, consisting of, or consisting essentially of high-conductivity few-walled CNTs dispersed thereon, wherein the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm. In certain embodiments, the CNTs comprise an intrinsic electrical conductivity in a range of about 1,000 S/cm to about 3,000 S/cm. In an alternative embodiment, the CNTs compromise an intrinsic electrical conductivity in a range of about 1,500 S/cm to about 2,500 S/cm.
Another aspect of the present disclosure provides a method of decreasing the level of chloride within a neuron comprising culturing the neuron on a substrate comprising, consisting of, or consisting essentially of high-conductivity few-walled CNTs dispersed wherein the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm. In embodiments, the CNTs comprise an intrinsic electrical conductivity in a range of about 1,000 S/cm to about 3,000 S/cm. In an alternative embodiment, the CNTs compromise an intrinsic electrical conductivity in a range of about 1,500 S/cm to about 2,500 S/cm.
Yet another aspect of the present disclosure provides a biocompatible implant comprising, consisting of, or consisting essentially of a substrate, the substrate comprising, consisting of, or consisting essentially of high-conductivity few-walled CNTs dispersed thereon, wherein the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm.
In some embodiments, the substrate further comprises the few-walled CNTs being suspended in a gum arabic solution. In certain embodiments, the CNTs suspended in gum arabic are coated homogenously on the substrate. In other embodiments, the substrate comprises the few-walled CNTs are not suspended in a gum arabic solution.
Another aspect of the present disclosure provides a method of treating or ameliorating injurious condition that is associated with elevated neuronal chloride in a subject comprising, consisting of, or consisting essentially of administering a biocompatible implant, the biocompatible implant comprising a substrate, the substrate comprising high-conductivity few-walled CNTs dispersed thereon, wherein the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm, to a subject in need of such treatment. In certain embodiments, the CNTs comprise an intrinsic electrical conductivity in a range of about 1,000 S/cm to about 3,000 S/cm. In an alternative embodiment, the CNTs compromise an intrinsic electrical conductivity in a range of about 1,500 S/cm to about 2,500 S/cm.
In some embodiments, the substrate further comprises the few-walled CNTs being suspended in a gum arabic solution. In certain embodiments, the CNTs suspended in gum arabic are coated homogenously on the substrate.
In other embodiments, the injurious condition is selected from the group of pain, epilepsy, traumatic neural injury, ischemia, stroke (cerebral ischemia) (Hershfinkel, et al. (2006) Nat. Neurosci. 12:725-727), brain edema (Kahle, K. T. et al. (2008) Nat. Clin. Pract. Neurol. 4:490-503), and neurodegenerative diseases including Alzheimer's disease (Lagostena, L. et al. (2010) J. Neurosci. 30:885-893) and psychosis (Hyde, T. M. et al. (2011) Neurosci. 31:11088-11095; Kalkman, H. O. (2011) Prog. Neuropsychopharmacol Biol. Psychiatry 35:410-414; Tao, R. (2012) J. Neurosci. 32:5216-5222), and combinations thereof.
Yet another aspect of the present disclosure provides a method of assessing KCC2 expression and/or assessing levels of chloride in a neuron found in a brain slice comprising, consisting of, or consisting essentially of placing the brain slice on substrate, the substrate comprising poly-di-methyl-siloxane (PDMS, polysil) that comprises conical indentation of at least 200 μm, high-conductivity few-walled CNTs dispersed thereon, wherein the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm.
Yet other aspects of the present disclosure provide a composition comprising the substrates described herein and a carrier. In some embodiments, the present disclosure provides kits comprising, consisting of, or consisting essentially of the composition, and/or carrier and instructions for use.
Yet another aspect of the present disclosure provides for all that is disclosed and illustrated herein.
The foregoing aspects and other features of the invention are explained in the following description, taken in connection with the accompanying drawings, wherein:
For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.
Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
One aspect of the present disclosure provides a substrate comprising, consisting of, or consisting essentially of high-conductivity few-walled CNTs dispersed thereon, wherein the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm. In certain embodiments, the CNTs comprise an intrinsic electrical conductivity in a range of about 1,000 S/cm to about 3,000 S/cm. In an alternative embodiment, the CNTs compromise an intrinsic electrical conductivity in a range of about 1,500 S/cm to about 2,500 S/cm.
Another aspect of the present disclosure provides a biocompatible implant comprising, consisting of, or consisting essentially of a substrate, the substrate comprising, consisting of, or consisting essentially of high-conductivity few-walled CNTs dispersed wherein the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm. In embodiments, the CNTs comprise an intrinsic electrical conductivity in a range of about 1,000 S/cm to about 3,000 S/cm. In an alternative embodiment, the CNTs compromise an intrinsic electrical conductivity in a range of about 1,500 S/cm to about 2,500 S/cm.
As used herein, the term “carbon nanotube” or “nanotube” or CNT means a structure at least partially having a cylindrical structure mainly comprising carbon and/or graphen of various composition. The nanotube includes single walled carbon nanotubes, double walled carbon nanotubes, few walled carbon nanotubes and multiwalled carbon nanotubes. The number of walls ranges from 1 to 100 and diameters range from 0.7 nm to 100 nm. Graphene includes graphene oxide, single layer graphene, few-layer graphene, reduced graphene. The term “few walled CNT” (fwCNT) refers to those CNTs having four or less walls. Examples of suitable carbon nanotubes, and method of making such nanotubes, are provided in U.S. Pat. No. 7,618,300, and U.S. Patent Application Ser. Nos. 61/127,711, 11/918,442, 10/759,592, 13/402,630 and Ser. No. 11/196,519, as well as Qi, H. et al. (2006) Chemistry of Materials 18:5691-5695, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the CNTs comprise an intrinsic electrical conductivity of at least 500 S/cm, 1,000 S/cm, 1,500 S/cm, 2,000 S/cm, 2,500 S/cm, 3,000 S/cm, 3,500 S/cm and 4,000 S/cm. In certain embodiments, the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm, in other embodiments the intrinsic electrical conductivity of the CNTs is at least 3,000 S/cm.
Substrates suitable for use in accordance with the present disclosure include conventional cell culture materials such as glass for in vitro applications, and biocompatible materials including, for example, polyimide, polyamide, polycarbonate, and silicone for in vitro and in vivo applications. In a preferred embodiment the substrate is polyimide, for example a polyimide membrane. Substrates having one or more extracellular matrix proteins disposed thereon in geometric patterns and dimensions described hereinabove can be made by micro- and nanofabrication methods known in the art. For example, bio-surface chemistry combined with micro contact printing by photolithography can be used to generate the combinatorial patterns to which a solution of the extracellular matrix proteins is added. The extracellular matrix proteins are selectively adsorbed by the micro-patterned regions to provide a substrate having a micro-patterned geometry coated with extracellular matrix proteins. Such methods are described for example by Agheli, H. et al. (2006) Nano Lett 6:1165-71, Seidlits, S. K. et al. (2008) Nanomedicine 3:183-199 and in the examples herein below. In addition, the substrates can be fabricated using nanomaterials such as nanowires, nanofibers and microwalled carbon nanotubes (MW CNTs). For example, substrates can be fabricated using MW CNT network patterns by applying CNT monolayer coatings to biocompatible polymer substrates such as polyimide, followed by selective adsorption of extracellular matrix proteins onto the CNT patterns. Such methods are described for example by Rao, S. G. et al (2003) Nature 425:36-37, Park, S. Y. et al. (2007) Adv. Mater. 19:2530-2534, and in the examples hereinbelow.
In some embodiments, the substrate further comprises the few-walled CNTs being suspended in a biocompatible solutions, such as gum arabic. In certain embodiments, the CNTs suspended in gum arabic are coated homogenously on the substrate.
The present disclosure is also based, in part, on the discovery that primary cortical neurons, when cultured on high-conductivity few-walled carbon nanotubes, showed a strikingly accelerated chloride shift caused by increased KCC2 expression. These findings suggest the integration of carbon nanotubes into neural engineering platforms aimed at injurious conditions of elevated neuronal chloride such as pain, epilepsy, traumatic neural injury and ischemia.
In one aspect, the present disclosure provides a method of upregulating kcc2 expression in a neuron comprising culturing the neuron on substrate comprising, consisting of, or consisting essentially of high-conductivity few-walled CNTs dispersed thereon, wherein CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm.
Another aspect of the present disclosure provides a method of decreasing the level of chloride within a neuron comprising culturing the neuron on a substrate comprising, consisting of, or consisting essentially of high-conductivity few-walled CNTs dispersed thereon, wherein the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm. In certain embodiments, the CNTs comprise an intrinsic electrical conductivity in a range of about 1,000 S/cm to about 3,000 S/cm. In an alternative embodiment, the CNTs compromise an intrinsic electrical conductivity in a range of about 1,500 S/cm to about 2,500 S/cm.
Yet another aspect of the present disclosure provides a method of normalizing the level of chloride within a neuron comprising culturing the neuron on a substrate comprising, consisting of, or consisting essentially of high-conductivity few-walled CNTs dispersed thereon, wherein the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm. In certain embodiments, the CNTs comprise an intrinsic electrical conductivity in a range of about 1,000 S/cm to about 3,000 S/cm. In an alternative embodiment, the CNTs compromise an intrinsic electrical conductivity in a range of about 1,500 S/cm to about 2,500 S/cm. The term “normalizing” means to bring back to baseline; or modification or reduction to the normal standard condition. (Taber's Cyclopedic Medical Dictionary 1309 (F.A. Davis Co., 18th ed. 1997)).
Yet another aspect of the present disclosure provides a method of treating or ameliorating injurious condition that is associated with elevated neuronal chloride in a subject comprising, consisting of, or consisting essentially of administering a biocompatible implant, the biocompatible implant comprising a substrate, the substrate comprising high-conductivity few-walled CNTs dispersed thereon, wherein the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm, to a subject in need of such treatment. In certain embodiments, the CNTs comprise an intrinsic electrical conductivity in a range of about 1,000 S/cm to about 3,000 S/cm. In an alternative embodiment, the CNTs compromise an intrinsic electrical conductivity in a range of about 1,500 S/cm to about 2,500 S/cm.
The term “biocompatible implant” includes micro-implants and nano-implants.
As used herein, the term “subject” is intended to include human and non-human animals. Exemplary human subjects include a human patient suffering from an injurious condition that is associated with elevated neuronal chloride. The term “non-human animals” includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and/or agriculturally useful animals (such as sheep, dogs, cats, cows, pigs, etc.), and rodents (such as mice, rats, hamsters, guinea pigs, etc.).
As used herein, the term “injurious condition that is associated with elevated neuronal chloride” refers to any condition that is characterized by, or presents as a symptom or biological effect, elevated neuronal chloride. In some embodiments, the injurious condition is associated with increased expression of KCC2. Such conditions include, but are not limited to, pain, epilepsy, traumatic neural injury, ischemia, stroke (cerebral ischemia), brain edema, and neurodegeneration including Alzheimer's disease and psychosis, and the like.
The term “administration” or “administering,” as used herein, refers to providing, contacting, and/or delivery of a biocompatible implant that comprises by any appropriate route to achieve the desired effect. These compounds may be administered to a subject in numerous ways including, but not limited to, orally, ocularly, nasally, intravenously, topically, as aerosols, suppository, etc. and may be used in combination.
Yet another aspect of the present disclosure provides a method of assessing KCC2 expression and/or assessing levels of chloride in a neuron found in a brain slice comprising, consisting of, or consisting essentially of placing the brain slice on substrate, the substrate comprising poly-di-methyl-siloxane (PDMS, polysil) that comprises conical indentation of at least 200 μm, high-conductivity few-walled CNTs dispersed thereon, wherein the CNTs comprise an intrinsic electrical conductivity of at least 2,500 S/cm.
The present disclosure also provides, in another embodiment, compositions comprising the substrates of the disclosure and a suitable carrier and compositions comprising the implants of the disclosure and a suitable carrier. The composition can be a pharmaceutical composition that contains a pharmaceutically acceptable carrier. The term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. The carrier in the pharmaceutical composition must be acceptable in the sense that it is compatible with the active ingredient and capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate.
The present disclosure also provides kits for using the substrates provided herein and treatment of injurious conditions that is associated with elevated neuronal chloride. Such kits include at least a first container containing a composition comprising the substrate described above in a carrier. The kits may additionally contain solutions or buffers for affecting the delivery of the first composition. The kits may further contain additional containers which contain compositions comprising further agents for treatment of neurodegenerative disorders and neurological injuries including for example, a drug for neural therapy, an anti-inflammatory agent, anti-apoptotic agent, or growth factor. The kits may further contain catheters, syringes or other delivering devices for the delivery of one or more of the compositions used in the methods of the invention. The kits may further contain instructions containing administration protocols for the therapeutic regimens.
The following examples are offered by way of illustration and not by way of limitation.
To determine whether exposing primary CNS neurons to CNT matrices could alter their gene expression so that their cell- and network physiological properties could be enhanced, neuronal chloride regulation in response to CNT matrix was assessed using few-walled CNT (fwCNT). FwCNT have simplified synthesis protocols and exceptional purity as compared to single-walled CNT. Feng, Y. et al. (2008) ACS Nano 2:1634-1638; Hou, Y. et al. (2009) ACS Nano 3:1057-1062; Qi, H., Qian, C., Liu, J. (2006) Chemistry of Materials 18:5691-5695; Qian, C. et al. (2006) J. Nanosci Nanotechnol 6:1346-1349. FwCNT are also favorable to multi-walled CN because fwCNT have lower defect density and higher electric conductivity.
Growth of Carbon Nanotubes and Silicon Oxide Nanowires (SiOx):
Few-walled carbon nanotubes (fwCNTs) were grown using a catalytic chemical vapor deposition (CVD) method. Co/Mo catalyst supported on porous MgO powder was used as the catalyst for nanotube growth and carbon monoxide (CO) was used as the carbon precursor. Qi, H., Qian, Liu, J. (2006) Chemistry of Materials 18:5691-5695. In a typical synthesis procedure, the as-prepared catalyst powder was placed in a horizontal tubular furnace (3 inches) and heated 950° C. Then CO was infused to the growth chamber for 30 minutes. The as-grown CNT product was then oxidized at 570° C. in air-argon mixture to remove amorphous carbon impurities produced during growth. Next, the material was boiled in HCl (5
Purity Analysis of fwCNT Preparation with X-Ray Photoelectron Spectroscopy (XPS):
To analyze purity of the fwCNT preparation, XPS, a spectroscopy surface chemical analysis technique that can quantitatively acquire the elemental composition, chemical and electronic state of the elements contained in a material was utilized. This method is characterized by great sensitivity. As-prepared fwCNT films were measured in an X-ray photoelectron spectrometer, model Axis Ultra (Kratos Analytical, Manchester, England), following the instructions of the manufacturer, followed by processing of the primary spectra with CasaXPS software (Casa Software, Teignmouth, England).
Preparation of Gum Arabic (GA) Solution, GA-Coating and Thin Films:
In order to improve aqueous solubility, biocompatibility with and adherence of neural cells and tissue, nanotubes were suspended in gum arabic (GA), a natural gum from hardened sap of Acacia senegal trees. Bandyopadhyaya, R. et al. (2002) Nano Lett 2:25-28. GA, a complex mixture of polysaccharides and glycoproteins has excellent biocompatibility as evidenced by its edibility. GA, a complex mixture of polysaccharides and glycoproteins has excellent biocompatibility as evidenced by its edibility. GA aqueous solution was prepared by adding GA (5 mg; Laboratory Grade, Thermo Fisher Scientific, Waltham, United States) in deionized water (100 mL) and stirring for 20 min. Dried-pure fwCNT (0.7 mg) was added to the GA solution and sonicated for 1 h. The fwCNT-GA solution was then centrifuged (7,200 rpm or 4,400 G) in an IEC Centra MP4 centrifuge for 2 hours to remove aggregates. The weight ratio of fwCNT to GA was 0.2. The same procedure was applied for solubilizing SiOx. Filtration of the fwCNT-GA solution through a polycarbonate membrane filter (pore size: 0.4 μm; HTTP02500, Millipore, Billerica, United States) yielded the GA-filtrate control reagent.
The spray coating method was used to prepare uniform thin films of fwCTN and SiOx nanowires on cover slips and cell culture dishes. For additional control experiments, the same cell-culture substrates were coated with gold films (100 nm) by E-beam evaporator (CHA Industries Solution, Fremont, United States). Finally, for all substrates, poly-
High-quality/high-purity fwCNTs were obtained that showed an extraordinary intrinsic electrical conductivity of 3,000 S/cm (
A suitable source of neural cells was used to address the question in primary neurons derived from the developing cerebral cortex of late embryonic rats. Yeo, M. et al. (2009) J. Neurosci 29:14652-14662. Their culture on fwCNT, prepared with a final coating of poly-D-lysine, sustained vital primary cultures with robust formation of neural processes, enlarged somata, increased expression of neuronal marker β3-tubulin, and notably the absence of any signs of cytotoxicity-neurotoxicity and biological non-compatibility in the cultures (
SEM confirmed enlarged soma size. Moreover, SEM showed the neurons in intimate proximity to the fwCNT matrix (
To determine whether the chloride shift in rat primary cortical neurons was accelerated, directed expression of a genetically-encoded chloride indicator, Clomeleon, was used to detect reduction of neuronal chloride.
Cortical Neuron Culture.
The preparation of primary cortical neurons was adapted from a previous protocol. Yeo, M. et al. (2009) J. Neurosci 29:14652-14662. Briefly, cortices were microdissected from embryonic rats (E18) or mice (E16.5). Rats provide an appropriate animal model and studies performed with rat primary neurons are representative and suggestive of results for human neurons.
The tissue was dissociated using papain, followed by mechanical dissociation. Cytosine arabinoside (2.5 μM) was added to cultures to inhibit the proliferation of non-neuronal cells. Cell suspension was plated at a density of 106 cells mL−1 onto tissue-culture dishes. 12 mm-diameter matrix-covered glass coverslips were contained in tissue culture dishes, typically n=3. Cortical neuronal culture prepared by this method yielded a majority population of neuronal cells, with negligible glial contamination, as evidenced by the absence of astrocytic protein, GFAP by Western blotting. Yeo, M. et al. (2009) J. Neurosci 29:14652-14662. Neuronal viability and differentiation were ascertained microscopically before, during and after experiments, and no evidence of neurotoxicity of the fwCNT preparation was obtained.
All animal procedures leading to primary cells and organotypic cultures, as used in this study, were performed with approval of the Duke University Animal Care and Use Committee under a valid institutional animal protocol, and in observance of National Institutes of Health guidelines.
Chloride Imaging.
Clomeleon-based ratiometric chloride imaging was conducted as described previously (Kuner, T., Augustine, G. J. (2000) Neuron 27:447-459; Yeo, M. et al. (2009) J. Neurosci 29:14652-14662), taking advantage of the ratiometric fluorescent chloride indicator protein, clomeleon, after appropriate calibration reactions in primary cortical neurons.
Statistical Assessment:
All results were expressed as mean±SEM. Two-tailed student's t-test or one-way ANOVA with post-hoc Tukey analysis were applied to ascertain statistical significance with p<0.05 indicating significant differences.
In rat primary cortical neurons, the chloride shift was accelerated (
To address whether the accelerated chloride shift that was evoked by fwCNT matrix was caused by KCC2 up-regulation, immunocytochemistry was used to determine KCC2 protein expression. Robust KCC2 up-regulation was indeed detected (
The reduction of neuronal chloride demonstrated in
Fluorescence Imaging of Active Presynaptic Terminals:
Fluorescence imaging of active presynaptic terminals was conducted as described previously. Li, H. et al. (2007) Neuron 56:1019-1033. In brief, cultured cortical neurons were exposed for 1 min to 50 mM KCl, 25 μM FM1-43 (Invitrogen), washed and left for 30 min. A micrograph of the live cells was obtained on an Olympus BX61 upright microscope, using a 40×/0.8 NA immersion objective, followed by a second 50 mM KCl exposure (1 min), and acquisition of a second micrograph, which was subtracted from the previous one. Betz, W. J., Mao, F., Bewick, G. S. (1992) J. Neurosci. 12:363-375. Puncta with bright fluorescence were recorded as morphological substrates of individual presynaptic terminals. (
To determine whether L-type voltage-gated calcium channels (VGCC) are involved in the chloride shift (Ganguly, K. et al. (2001) Cell 105:521-532; Yeo, M. et al. (2009) J. Neurosci 29:14652-14662) their functional expression was verified using a fluorescently-labeled compound that directly binds to VGCC, bodipy-DM-dihydropyridine.
VGCC Receptor Binding Studies.
Fluorescence imaging of dihydropyridine binding to L-type VGCC was conducted following previous reference. Schild, D., Geiling, H., Bischofberger, J. (1995) J Neurosci Methods 59:183-190. Briefly, 1 μM Bodipy-DM-DHP (Invitrogen, Carlsbad, United States) was applied to primary cortical neuronal cultures on for 1 h (37° C.), cells were washed and fixed in 4% paraformaldehyde for 20 min, mounted on glass-slides using fluoromount, and imaged, using either green- or red filter settings. Quantitative assessment was conducted following previous reference (Yeo, M. et al. (2009) J. Neurosci 29:14652-14662), using ImageJ.
Immunocytochemistry:
Confocal fluorescence imaging was conducted after immunolabeling for β3-tubulin (mouse monoclonal antibody; 1:200, Iowa hybridoma bank, Iowa City, United States), and KCC2 (rabbit polyclonal antibody; Abcam; 1:200). Id.. Fluorescently labeled sections were visualized in a Zeiss LSM710 (Carl Zeiss AG, Oberkochen, Germany) confocal imaging suite with lasers tuned to the emission spectra of the secondary fluorescent antibodies (coupled to Alexa-488 and Alexa-595 dyes), as described previously. Li, J. et al. (2011) Environ Health Perspect 119:784-793.
Wide-field fluorescence microscopy was conducted after immunolabeling for VGCC isoforms Cav1.1, 1.3 and 1.4. Labeling for Cav1.2 was not conducted because preliminary testing revealed absence of a PCR product for Cav1.2, whereas all other isoforms could be detected in fwCNT-cultured neurons (primers and conditions available upon request). The following primary antibodies were used: mouse anti-Cav1.1 (Cat# MA3-920; 1:100, Thermo Fisher Scientific, Waltham, United States), mouse anti-Cav1.3 (Clone# N38/8; 1:100, NeuroMab, Davis, United States), and rabbit anti-Cav1.4 (Cat#: LS-C94032; 1:100, LS Biosciences, Seattle, United States). Secondary fluorescent detection antibodies as described above “Confocal Imaging.” Micrographs were acquired on an Olympus BX61 upright microscope using a 40× Olympus objective (Olympus, Center Valley, United States), connected to Roper high-resolution CCD-camera with ISEE software (Roper Scientific, Inc., Trenton, United States).
Scanning Electron Microscopy:
SEM was conducted according to previous reference. Rak, K. et al. (2011) J Biomed Mater Res A 97:158-166. In short, the samples were dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, 100%) and then dried with hexa-methyl-disilazane. The samples were sputter-coated with gold using a Denton Desk IV system. (Denton Vacuum, LLC, Moorestown, United States). Samples were imaged using a FEI XL30 FE-SEM.
Transmission Electron Microscopy:
TEM was conducted according to previous reference, (Kesty, N. C. et al. (2004) Embo J 23:4538-4549) with the following modification to accommodate culture of primary cortical neurons on poly-
Morphometry for confocal and light-microscopy acquired images was conducted as previously described. Yeo, M. et al. (2009) J. Neurosci 29:14652-14662.
FwCNT-cultured primary cortical neurons expressed VGCC at dramatically increased levels of DHP-binding vs. control (
These findings prompt the question how a rise in intracellular calcium leads to increased expression of Kcc2. Based on known interactions in central neurons between increased intracellular calcium, calmodulin and calcium/calmodulin-dependent kinase II (CaMKII), [15] tests were conducted to determine whether CaMKII activity is required for the accelerated chloride shift and KCC2 upregulation when culturing on fwCNT matrix. This novel concept was confirmed using specific inhibitors of CaMKII, but remarkably only for fwCNT-, not for Au matrix-cultured neurons (
These findings originated from cortical neurons maintained as primary cultures, which recapitulate the chloride shift as it happens in perinatal development of the vertebrate CNS.
To determine the properties of fwCNT matrices in their natural surroundings, which include a layered cortical architecture with neural connectivity as a result of in vivo development, mice brain slice cultures were analyzed.
For this purpose, mice were engineered so that a 2,500 base-pair proximal promoter fragment of the Kcc2b gene drives a red-shifted luciferase (red LUC) reporter gene. This construct was genomically integrated, by homologous recombination in mouse embryonic stem cells, into the inert Rosa26 locus, giving rise to otherwise normal and fertile mice, which transmitted the engineered mutation (
Mouse Gene Targeting of the Rosa26 Locus with Kcc2-Promoter LUC Reporter Construct:
A plasmid, pKcc2-red Luc that contains a mouse 2.5 kb Kcc2b (fore-brain specific Kcc2 isoform) promoter DNA fragment downstream of a polyA+ cassette to protect against cis-acting promoter/enhancer interference of the Rosa26 locus was constructed. Eggermont, J., Proudfoot, N. J. (1993) Embo J. 12:2539-2548. This cassette containing the polyA+-Kcc2-promoter DNA was subcloned upstream of the red luciferase coding region in a vector called pBasicRedLuc, harboring a codon-optimized Italian firefly (Luciola italica) luciferase (Genetargeting Systems). Pad and AscI restriction sites were added at the 5′ and 3′ ends of the final construct to enable cloning into a modified Rosa26 targeting vector, pROSA26Am1. Srinivas, S. et al. (2001) BMC Dev Biol 1:4. The resulting construct was linearized with MfeI, purified and electroporated into embryonic stem (ES) cells, strain R1. PCR screening of G418-resistant ES-cell colonies was used to identify homologous recombination into the Rosa26 locus. For that purpose one primer was designed upstream of the short homology arm of the Rosa26 targeting vector. This primer (R26F) had the sequence: 5′-CCTAAAGAAGAGGCTGTGCTTTGG-3′.
Another primer (KCC2R) was designed within the 5′ end of the Kcc2 promoter and had the sequence: 5′-CTTATCCTTGAGAGACGTACTAGTCC-3′.
The 1.3 kB PCR product amplified from genomic DNA of homologously recombined ES-cell clones was identified for 11 clones of a total of 48 clones screened, indicating correct, orthotopic targeting. Three such clones were expanded and verified again. Recombinant ES-cells from these clones were used for microinjection into blastocysts to generate chimeric mice. Breeding of these chimeras to wildtype C57B16 mice established germline transmission of the knockin and established the mouse line as used here.
Organotypic Culture:
Cortical slices were cut and then cultured from neonatal mice using a method described previously. Pond, B. B. et al. (2006) J. Neurosci 26:1396-1406. In brief, brains were removed from euthanized Kcc2 red LUC mice into complete HBSS. The brains were then embedded in low-gelling temperature agarose (2-3% w/v). Coronal slices (300 μm) were cut from the agarose block with a Leica VT1000S vibratome. The slices were then transferred onto cell culture inserts (PICMORG50; Millipore, Billerica, United States) in culture dishes (35 mm) filled with growth medium (BMEM, 10% bovine calf serum, 1 m
After 3 days in culture, bioluminescence from cultured slices was determined with a cooled CCD camera (IVIS100; Xenogen, Alameda, United States). After removing PDMS sheets and obtaining a baseline, luciferin (20 μl; 500 μm) was dropped onto each slice and bioluminescence was measured afterwards. Bioluminescence reached a plateau within 5 min. Images were acquired at time-point 10 min after luciferin application with 5 min exposure
For qRT-PCR assays, cortical regions exposed to PDMS sheets were dissected on the cell culture inserts, transferred into centrifuge tubes, then quick-frozen for subsequent total RNA extraction.
For KCC2 immunolabeling, brain slices were immersion-fixed in paraformaldehyde (2%) for 2 h, then immunolabeled with KCC2-specific antibody as described under Immunocytochemistry.
qRT-PCR:
Total RNA from cortical neurons was extracted and quantified as previously described. Li, J. et al. (2011) Environ Health Perspect 119:784-793. Prior to reverse transcription, total RNA was subjected to DNaseI treatment (Invitrogen, Carlsbad, United States) to eliminate genomic DNA. DNaseI-treated total RNA (1 μg) was then subjected to first-strand cDNA synthesis with Superscript-III reverse transcriptase (Invitrogen, Carlsbad, Untied States). qPCR was performed using a ABI 7900 RT-PCR platform. First-strand cDNA (˜100 ng or 2 μl of a 20 μl RT reaction) was processed using SYBR-Green PCR Mastermix (Qiagen, Venlo, Netherlands). Each reaction was performed in triplicates. The following primers (mouse sequence) were used in qRT-PCR:
The relative increase in reporter fluorescent dye emission was monitored in an ABI quantitative real-time thermocycler platform. The level of Kcc2 or red Luc mRNA, relative to β3-tubulin, was calculated using the ΔΔCt method, where Ct was defined as the number of the cycle in which emission exceeded a pre-set threshold.
First, results unambiguously demonstrates generation of functional reporter, luciferase, controlled by a 2.5 kB DNA sequence of the proximal promoter fragment of the Kcc2 gene, in primary cortical neurons cultured from these mice. Second, it suggests common principles of transcriptional regulation shared by the 2.5 kB fragment and the endogenous Kcc2b promoter.
fwCNT PDMS Device Preparation:
For exposure of cultured brain slices, semi-flexible devices made of poly-di-methyl-siloxane (PDMS, polysil), using molds with customized conical indentations of >200 μm length, so that 250 μm thin brain slices were exposed throughout their depth were generated. The PDMS substrates with pillars were fabricated by the standard molding procedures used previously in soft lithography for fabrication of PDMS stamps. Kumar, A., Whitesides, G. M. (1993) Applied Physics Letters 63:2002. A sheet of aluminum foil (0.25 mm) punched with over-the-counter quilting needles (size 7) was used as the replica mold. The elastomer and curing agent mixture (Sylgard 184 Silicone Elastomer Kit, Dow Corning, Midland, United States) was cast on the mold. After degassing and curing, the pillared PDMS stamp was removed from the mold surface. The PDMS substrate was irradiated in a plasma sterilizer for 5 min to render the surface hydrophilic before fwCNT coating. Then the fwCNT-GA solution was spray-coated on the PDMS substrate. SEM images of the as-prepared PDMS stamps with pillars protruding from the surface were acquired at 30° tilt of the sample stage (
The PDMS casts were coated with GA-solubilized fwCNT, finally with poly-
A dramatic acceleration of a fundamental neural maturation mechanism by a fwCNT-matrix is driven by gene regulatory changes, namely upregulation of Kcc2. This process involved transcriptional de-repression, as demonstrated for physiological in vitro development. Yeo, M. et al. (2009) J. Neurosci 29:14652-14662. Mechanistically, a direct contact of fwCNT matrix to the neuronal outer plasma membrane in primary neuronal culture was verified. FwCNT are highly conductive, so that “external wiring” of single neurons will enhance their electrical activity cell-autonomously. Extraordinarily high electrical conductivity of the fwCNT culture matrix and direct interfacing of the neuronal plasma membrane with fwCNT will also promote neuron-to-neuron connections, in addition to biological neuron-to-neuron connectivity based on direct proximity of somata and processes of individual neurons. Therefore, the observed novel effect of fwCNT can be regarded as based on two major principles. One is the increased electrical conductivity of the matrix—similar in principle to the control Au-matrix (even though conductivity of Au is substantially increased over that of the used fwCNT). In addition, different from Au, is the “external wiring” of the nerve cell, cell-autonomously and neuron-to-neuron, by an electrically conductive nanomaterial. The joint effect will be increased electrical activity of the neurons. Increased activity translates into increased functional expression of VGCC, which promotes Kcc2 expression via calcium influx. CaMKII is involved in the accelerated chloride shift, whereby CaMKII provides the link between calcium influx via VGCC and subsequent transcriptional activation of Kcc2. More relevant for the present study, VGCC activity is critical for the accelerated chloride shift, as critical as CaMKII, since their respective selective block eliminated the accelerated chloride shift (concept summarized in
The mechanism outlined for primary neuronal cell culture could also occur in brain slices, yet in this platform awaits more in-depth study and definitive proof in future studies.
The documented acceleration of the chloride shift via interfacing with fwCNT could form the basis for their use as an advantageous and innovative tool to precisely control cell-physiological properties of CNS neurons, which in turn dictate neural circuits' functions. KCC2 has recently been characterized as neuroprotective. Pellegrino, C. et al. (2011) J. Physiol 589:2475-2496. This means that approaches that can upregulate KCC2, such as direct exposure to fwCNT, will have a neuroprotective effect. Future use of fwCNT-coated devices can now be envisioned in conditions of neural injury that have been associated with Kcc2 down-regulation and increased neuronal chloride.
FwCNT matrix functions in a neuro-protective manner with respect to chloride upregulation in response to neural injury that is mediated by air-blast, a model for neural injury by explosions, and axotomy, a direct traumatic injury of the nerve cell where its axonal process is cut-off (or amputated at the cellular level) (
These results were obtained with rat primary cortical neurons cultured on fwCNT matrix, then subjected to the two modalities of neurotrauma (
Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.
This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 61/616,027 filed on Mar. 27, 2012, which is incorporated herein by reference in its entirety.
This invention was produced in part using funds from the Federal Government under NIH Grant No.: R21 NS066307 entitled “Sex-specific gene regulation of neuronal chloride co-transporter, Kcc2” and NSF Cooperative Agreement Number: EF-0830093. Accordingly, the Federal Government has certain rights to this invention
Filing Document | Filing Date | Country | Kind |
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PCT/US13/32325 | 3/15/2013 | WO | 00 |
Number | Date | Country | |
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61616027 | Mar 2012 | US |