Patent Application
20140295451
The invention is related to a method of visualization and characterization of the nervous system and, more specifically, of the central nervous system, by staining the same histological section for metal impregnation and immunocytochemistry. The doubly stained histological sections are subsequently subjected to a qualitative and quantitative morphological and antigenic and/or neurochemical analysis, by means of conventional optical microscopy or confocal laser scanning microscopy.
The histological method of metal impregnation by applying potassium dichromate and silver nitrate developed by Camillo Golgi in 1873 continues, with all its subsequent variations, to still represent a method of reference today, and one essential for the histological analysis of the nervous tissue and, more specifically, of the central nervous system. At present the most used metal impregnation is the one proposed by Cox in 1891, known as mercuric impregnation or Golgi-Cox's method or black reaction, which foresees replacement of silver nitrate with mercuric chloride. This technique is widely used for the qualitative analysis of neuron morphology and in determining the quantity of dendritic spines, and the length and complexity of the dendrite branching.
Another successful technique applied to the visualization and characterization of tissues is immunohistochemistry, a technique which exploits the ability of antibodies labeled with suitable markers, preferably fluorescent, to bind specific antigens with high affinity. This technique is widely used to localize antigens on both a cellular and sub-cellular level, and it is widely applied on the central nervous system as well.
Both of these two methodologies of visualization and characterization of tissues have in recent years been improved by applying confocal laser scanning microscopy (CLSM) using reflection mode, the latter a technique suitable for the modeling and reconstruction in 3D of cellular structures, especially neurons. The very extensive use made recently of confocal laser scanning microscopy applied to these techniques is due to the possibility of obtaining top-quality images from specimens prepared for examination under conventional or fluorescent light. Confocal reflection microscopy, in fact, allows, in the case of the mercuric impregnation technique, to visualize the tissues impregnated with the metal particles in an advantageous manner, because the metal particles reflect the laser light and do not display a photo-bleaching phenomenon. In the case, instead, of immunohistochemistry, this analytical method for tissues allows to control field depth, to reduce background noise, and to collect serial optical sections from the specimen. Furthermore, confocal laser scanning microscopy allows to survey fine details which can represent substantial information and which would be lost with conventional microscopy.
In the past, some attempts have been made to combine metal impregnation and labeling with the immunoperoxidase enzyme. In this regard, the Golgi impregnation methods after retrograde transport of the peroxidase, initially proposed by Somogyi et al. and Freund and Somogyi, respectively applied to the visual cortex of a cat (Somogyi P et al., 1983, Neuroscience, 9, 475-490) and of a monkey (Freund T F, Somogyi P, 1983, Neuroscience 9, 463-474), can be remembered. However, the methods proposed are considerably complex and poorly reproducible.
Techniques have also been proposed for tissue perfusion and fixation that are such so as to make Golgi's method and its variations more versatile, depending on the experimental aims. With regard to fixation, several times have been proposed for the fixation of tissues in formalin, from a few hours (Williams R S, 1983, J. Neuropath. Exp. Neurol. 42, 210-212) to several years (Goradz R et al., 2003, J. Neuroscience Meth. 131, 1-7).
In no case, instead, the combination of double staining with the Golgi-Cox mercuric impregnation method and immunofluorescence has been proposed. This is presumably due to the fact that the metal impregnation reaction and immunofluorescence are deemed incompatible, due to the significant differences in the perfusion and fixation procedures foreseen for the two techniques (Lee K W et al., 2006, Proc. Natl. Acad. Sci. USA 28, 103, 3399-3404).
Therefore, to remedy the drawbacks of the methods in use, a purpose of this invention is to develop a method of visualization and characterization of nervous system tissues and, more specifically, of the central nervous system, by staining the same histological section for metal impregnation and immunocytochemistry. In particular, the purpose of this invention is to develop a method simple to perform and highly reproducible, which allows scientists to simultaneously obtain an amplification of the effects of the two methods for visualization of the morpho-structural conformations of nervous tissues and a synergism between them, allowing them to analyze the same histological sections by means of conventional optical microscopy or confocal laser scanning microscopy.
The method described hereinafter is, therefore, based on combining the two histological staining techniques in the same tissue specimens to be analyzed, that is, metal impregnation and immunohistochemistry, associated to the analysis of the specimens thus prepared with conventional optical microscopy or, preferably, with confocal laser scanning microscopy.
The solution found to overcome the incompatibility among the different metal impregnation procedures, with special regard to Golgi-Cox impregnation but not limited to this metal impregnation, and immunohistochemistry, essentially consists in the fixing procedures of the tissues to be studied, which foresees fixation of tissues ex vivo using appropriate concentrations of paraformaldehyde, optionally pre-fixed with the same ex vivo fixing solution comprising paraformaldehyde.
Therefore, the object of this invention is a method of visualization and characterization of the nervous system by combining staining for metal impregnation and immunocytochemistry, comprising at least the steps of:
The method according to the invention finds application in the experimental and clinical field for the study of the characteristics of neuronal connections in the central nervous system.
The method and its possible embodiments, as also its advantages, will be made clearer by the detailed description of the invention which follows, with the aid of the figures annexed to it.
As known and as already noted previously, Golgi's metal impregnation technique, including the variant of Golgi-Cox's mercuric impregnation, and the immunofluorescence technique cannot be applied in the same tissue section. This incompatibility essentially resides in the fact that for immunohistochemistry, the critical point is the step of tissue fixation, since denaturation of the sites for recognition of the antibodies and the diffusion of proteins from their initial localization must be prevented for avoiding the production of experimental artifacts and accordingly poor reliability of the experimental data.
To solve this technical problem, the inventors have developed a method which foresees fixation of the tissues ex vivo using aqueous solutions of paraformaldehyde in suitable concentrations, optionally pre-fixed with the same solution comprising paraformaldehyde. Of these two fixation steps the second, that is, the ex vivo fixation, is the critical one for the immunohistochemical staining, because for the staining of the tissues to be adequate, the antibodies have to penetrate in the entire depth of the tissues and to bind the specific antigens, in spite of the protein's fixation. The conditions for carrying out of this fixation are therefore essential to obtain an effective immunochemical staining. In this respect, although it is known that the protein fixing process with formaldehyde is slow and just about complete only in 24 hours (Helander K G, 1994, Biotech. Histochem. 69, 177-179), it is not foregone that the latter will at the same time also allow an efficient penetration of the antibodies necessary for the immunohistochemistry and their actual binding with the antigens.
For the method of visualization and characterization of the nervous system by combining the staining for metal impregnation and immunocytochemistry, which is the object of the invention, the fixing step is therefore such to allow both fixation of the proteins and an efficient penetration of the antibodies for immunohistochemistry. More specifically, the fixation of nervous system specimens foresees a fixation of a tissue specimen to be examined with an aqueous solution comprising paraformaldehyde in a concentration between 1 and 2% w/v or a paraformaldehyde 4% w/v in Sorensen's Phosphate Buffer (PBS 0.4M, pH 7.4).
This method can, as a matter of fact, be carried out according to two different protocols, or rather, two different embodiments of the same method, which in any case foresee the fixation of specimens to be examined by immersion thereof at a temperature of 4° C. for at least 8 hours to up to 12 h in a paraformaldehyde solution 4% in PBS (0.4M, pH 7.4) followed by metal impregnation or in a composition comprising both the fixative, that is, the paraformaldehyde in concentration between 1 and 2% w/v, and the reagents for the metal impregnation, preferably according to the known Golgi-Cox method.
In the case, in which the fixation is carried out with an aqueous solution comprising paraformaldehyde between 1 and 2% w/v and the reagents for metal impregnation, the metal impregnation step is already started in the fixation step, and the subsequent impregnation step is carried out to renew the reagents and maximize impregnation. Essentially, in this second case also the fixative properties of the salt solution are exploited for the preferred Golgi-Cox mercuric impregnation, fixative properties which are moreover the basis for the metal impregnation itself, combined with the fixative properties of the paraformaldehyde. The composition that can be used for the combined fixation and metal impregnation step consists in an aqueous solution having pH 7.4 comprising:
Optionally, the step of fixation with paraformaldehyde 4% or with the composition comprising paraformaldehyde between 1 and 2% and the reagents for metal impregnation can be preceded by a pre-fixation by perfusion with the same solution comprising paraformaldehyde of the subsequent step of fixation ex vivo.
This perfusion and pre-fixation step is not, however, essential to achieve the purpose of the invention and can therefore be omitted, thus allowing the application of the method also in the clinical field.
In relation to the metal impregnation technique and immunohistochemistry, in both protocols these are performed according to modes known to an expert in the field. Therefore, as known, the mercuric impregnation can be obtained by:
The immunohistochemistry, instead, is carried out on sections of impregnated tissue specimens and then, after metal, preferably mercuric, impregnation, the method comprises the further step of cutting the specimens in slices 50-100 μm thick with a cryostat or a vibratome. The sections obtained are then subjected to the following treatments before the immuno-staining in free floating with the suitable reagents:
In the case that the tissue is cut in slices with a cryostat, it must be treated before the slicing with a cryo-protective saccharose aqueous solution 30% by immersion for two or three days. This step can be skipped if a vibratome is used.
Furthermore, when cutting with a cryostat, the temperature of the specimen is preferably of −4° C. and the temperature of the blade −14° C. In any case, the slices are collected in pits that contain H2O.
Furthermore, as known to an expert, the method according to the invention can comprise further steps, for example, a wash to remove the traces of fixatives used for the mercuric impregnation and for immunohistochemistry.
Typically, therefore, for what mentioned previously, according to an embodiment applicable in the experimental field, and when a greater quality of the visualization deriving from the immunohistochemistry is pursued, the method according to the invention comprises the steps of:
The embodiment of the method according to the invention without the pre-fixation of the tissue can be applied in clinical field.
In both cases, as known to an expert in the field, further steps must also be foreseen in between the main fixation steps, the metal impregnation and immunohistochemistry previously detailed. These steps are:
From the experimentation carried out, it has been possible to verify that the method of visualization and characterization of the nervous system, according to the invention, is in practice a simple method, easily reproducible which gives high-quality results and, therefore, fulfilling the established purposes.
The main advantages offered by this method can be summarized as follows:
For the research described hereinafter, Sprague-Dawley male albino rats, supplied by Charles River (Como, Italy) with a weight of 200-225 g, have been used. All the experiments have been performed following the guidelines of the U.E. which regulates the use of experimental animals (CEE N °86/609) and the guidelines for the care and use of laboratory animals approved by the Italian Society for Neuroscience. Before any treatment, the animals were deeply anesthetized with urethane (1.3 g/kg) and subjected to transcardial perfusion with a physiological solution (saline solution 0.9%) or with Sorensen's Phosphate Buffer (PBS) 0.4M.
The metallic impregnation has been carried out with a Golgi-Cox solution composed by: 5% potassium dichromate, 5% mercuric chloride, 5% potassium chromate (pH 6.5) (Glaser and van der Loos, 1981, ref. cit.).
The method of double staining by Golgi-Cox mercuric impregnation and immunohistochemistry has been validated on the “meso-cortico-limbic” and “nigrostriatal” nervous pathways that contain neurons and tyrosine hydroxylase (TH) positive fibers. To this end, the following reagents have been used: commercial primary poly and monoclonal antibodies of rabbit, and anti-tyrosine hydroxylase (TH) mice, anti post-synaptic-density 95 (PSD-95), and anti-synapsin I (SynI), respectively supplied by Santa Cruz Biotechnology Inc. (poly-clonal rabbit antibodies anti-TH (r_anti-TH) and anti-SynI; monoclonal mice antibodies anti-PDS95) and Sigma-Aldrich (monoclonal mice antibodies anti-TH (m_anti-TH)). The secondary antibodies used were: anti-mice IgM biotinylated goat and fluorescein-streptavidin (Vector Laboratories, Burlingame, Calif.), anti-rabbit Alexa Fluor 546 (1:200, Molecular Probes).
As a control procedure, three rats were deeply anesthetized and subjected to transcardial perfusion with a saline solution 0.9%, as above. The brains were immediately immersed in the Golgi-Cox solution, according to the traditional procedure, without any fixation and subsequent post-fixation with paraformaldehyde (Fregerslev S et al., 1971, Histochemie, 26, 289-304).
The fixation and staining protocol with mercuric impregnation and immunohistochemistry according to the method of the invention have been carried out according to the two ways of fixation ex vivo with paraformaldehyde 4% and mercuric impregnation in sequence or combined fixation and metal impregnation, described in detail hereinafter respectively in example 1 as method a) and in example 2 as method b).
Sprague-Dawley Charles River rats, (Como, Italy) with a weight of 200-225 were deeply anesthetized with chloral hydrate. Then, transcardial perfusion was carried out with 400 μl of physiological solution, followed by 200 μl of paraformaldehyde solution 4% in PBS (pH 7.4). The brains were carefully removed from the skull and placed in the same paraformaldehyde-based fixation solution at 4° C. for the entire night. The brains were then washed in PBS for at least 8 h (5 washes) to remove any trace of fixative.
Subsequently, each brain was immersed in the Golgi-Cox solution (in a quantity sufficient to entirely cover the specimens) and maintained in the dark for two days. The solution was then replaced with a fresh Golgi-Cox solution and the brain was maintained in it for a further 14 days, always in the dark.
The specimens were then washed with H2O (3×5 min.) and transferred for two or three days in a cryo-protective saccharose solution 30%. The brains were then cut in coronal slices 50-100 μm thick. With the cryostat, the optimal temperature of the specimen is −4° C. and the optimal temperature of the blade is −14° C. The slices obtained were collected in pits containing H2O and treated with an ammonia solution 30% for 40 min. in the dark at room temperature. The slices were rinsed with distilled water for 10 min and fixed for 10 min. with a photographic fixative (Kodak Fix for Paper) diluted 1:7. Finally, the slices were rinsed in distilled water (10 min.) and collected in PBS for the subsequent immuno-staining in free floating. For the immunohistochemical staining, all the slices were washed in PBS (3×10 min.) at room temperature and, to prevent non-specific bonds, the slices were pre-incubated in a solution containing: 5% normal goat serum, 5% bovine serum albumin (BSA), and 0.5% Triton-X-100 in PBS at 4° C. for the entire night. The slices were then incubated with the primary antibodies in the following combinations:
All the sections were washed 3×10 min. in PBS and then incubated with the secondary antibodies according to the following pattern:
At the end of the incubations with primary and secondary antibodies, all the sections were washed (3×10 min) in PBS, placed on a slide, and sealed with Vectashield (Vector Laboratories, Burlingame, Calif.).
Method b) is the method which foresees combining of the fixation with paraformaldehyde and metal impregnation and, for this method, the composition already described previously is prepared. The preparation of this composition is described hereinafter.
The following aqueous solutions are prepared separately:
Then, the different components are slowly mixed one with another and under continuous stirring as follows:
The pH of the solution thus obtained is controlled and, if necessary, it is brought to pH 7.4. The composition is then maintained in the dark for 5 days and then filtered.
The animals, the anesthesia, and the perfusion are as in example 1, with the difference that in this case, the pre-fixation by perfusion has been carried out with the composition comprising the paraformaldehyde and the reagents for metal impregnation prepared as described above. The perfusion with the composition has been followed by fixation of the brains ex vivo by immersion in the same composition. Continuation of the metal impregnation and immunofluorescence has been carried out as in example 1.
All the tissue specimens (examples 1 and 2) have been examined with conventional light microscopy to evaluate whether the impregnation was successful and with fluorescence microscopy and confocal laser scanning microscopy to evaluate whether the immunofluorescence reaction was successful. Both method a) of example 1 and method b) of example 2 according to the invention have been tested in the meso-cortico-limbic dopaminergic pathway, which contains several cerebral nuclei which go from the mesencephalon to the basal ganglia to the frontal cortex. In the mesencephalon (ventral tegmental area and substantia nigra) are the cellular bodies of the neurons containing dopamine (DA) which project their axons predominantly in the basal nuclei and in the frontal cortex. The expected results are, therefore, for the mesencephalon:
For the nigro-striatal area, instead, the expected results are:
It was first of all surveyed that the pre-fixation and fixation ex vivo with paraformaldehyde 4%, or the pre-fixation by perfusion and fixation ex vivo with the composition comprising both paraformaldehyde and the reagents for the mercuric impregnation of the cerebral tissues do not prove to have significant effects on the standard Golgi-Cox mercuric impregnation, nor on the immunohistochemical staining.
In fact, in all the preparations examined with the Golgi-Cox procedure, a good impregnation of the neural elements was obtained, which proved homogenously distributed in the tissue. As a rule, only a portion of the total number of neurons proved stained, equal to approximately 5-10%. These neurons, distributed randomly and uniformly, resulted complete with dendritic tree, dendritic spines, and axons. Occasionally, also some glial elements and the lumen of some capillaries were stained. In all the impregnated slices examined under a conventional white light microscope, the stained elements appeared of an intense black color on clear background. The immunohistochemistry staining of the tissues stained with Golgi-Cox displayed very luminous fluorescent elements and, as expected, the penetration of all the antibodies was approximately 25-40 μm in both sides of the slice. However, immunoreactivity was only found in the sections coming from those brains which were perfused and post-fixed with paraformaldehyde, while no fluorescence was obtained in those brains that were only perfused with saline solution and fixed with Golgi-Cox solution alone (test control rats).
In particular, in the mesencephalon, the neurons impregnated with Golgi-Cox and those TH-positive (fluorescence) were simultaneously visualized with the confocal laser scanning microscope (CLSM). These neurons proved to be, as is customary, distributed over all the tissue, and did not seem to have been negatively affected by the previous impregnation. As a matter of fact, in all the preparations, the neurons always resulted complete with dendrites and axons, and the immunoreactivity surveyed was always homogenous.
The distribution of the immunoreactivity to the PSD-95 in the mesencephalon resulted not to be homogeneous.
In the striatum with Golgi-Cox staining and immunohistochemistry for the TH, numerous impregnated medium spiny neurons (MSN) were observed and immersed in a dense net of TH-positive fibers (
Furthermore, the immunoreactivity with the PSD-95 and the SynI in the striatum was detected to be uniform. When the double immuno-staining was carried out with these antibodies in sections stained with the Golgi-Cox impregnation, it was possible to survey the relations between these two antigens in the dendrites and in the dendritic spines of the MSN (
In conclusion, the results obtained show that the method of visualization and characterization of the nervous system that is the object of the invention fulfills its purposes, since it has proven to be a simple and inexpensive method, suitable for both conventional microscopy and confocal microscopy, which grants the possibility to pair two techniques that are incompatible, that is, Golgi-Cox mercuric impregnation and immunohistochemistry. It has, instead, proven possible to combine these two techniques which have long been incompatible, for them to be used simultaneously in the same sections (Lee et al. 2006, ref. cit.), by following appropriate fixation procedures according to the method that is the object of the invention and, at the same time, giving optimal results extensively correspondent to the expected results based on the available knowledge in the field.
| Number | Date | Country | Kind |
|---|---|---|---|
| PD2011A000081 | Mar 2011 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2012/054454 | 3/14/2012 | WO | 00 | 10/8/2013 |
